In this paper, the development of a fully implantable wireless sensor able to provide continuous real-time accurate pressure measurements is presented. Surface Acoustic Wave (SAW) technology was used to deposit resonators on crystalline quartz wafers; the wafers were then assembled to produce a pressure sensitive device. Excitation and reading via a miniature antenna attached to the pressure sensor enables continuous external interrogation. The main advantages of such a configuration are the long term stability of quartz and the low power necessary for the interrogation, which allows 24/7 interrogation by means of a hand-held, battery powered device. Such data are of vital importance to clinicians monitoring and treating the effects of hypertension and heart failure. A prototype was designed and tested using both a bio-phantom test rig and an animal model. The pressure traces for both compare very well with a commercially available catheter tip pressure transducer. The work presented in this paper is the first known wireless pressure data from the left ventricle of the heart of a living swine.
SAW; Pressure sensor; Implantable; Patient monitoring; Telemetry; Telemedicine; Heart; Wireless
Cardiac myosin binding protein-C (cMyBP-C) is a multi-domain (C0–C10) protein that regulates heart muscle contraction through interaction with myosin, actin and other sarcomeric proteins. Several mutations of this protein cause familial hypertrophic cardiomyopathy (HCM). Domain C1 of cMyBP-C plays a central role in protein interactions with actin and myosin. Here, we studied structure-function relationship of three disease causing mutations, Arg177His, Ala216Thr and Glu258Lys of the domain C1 using computational biology techniques with its available X-ray crystal structure. The results suggest that each mutation could affect structural properties of the domain C1, and hence it’s structural integrity through modifying intra-molecular arrangements in a distinct mode. The mutations also change surface charge distributions, which could impact the binding of C1 with other sarcomeric proteins thereby affecting contractile function. These structural consequences of the C1 mutants could be valuable to understand the molecular mechanisms for the disease.
As waiting lists for lung transplantation are ever increasing, the number of organ donors is not able to keep pace with it. Living donor lobar lung transplantation is a source of organs which could be lifesaving in end-stage lung disease patients who cannot wait for cadaveric organs due to deteriorating lung function and clinical condition. Two young women with end stage cystic fibrosis received lobes from their relatives and an altruistic friend. They are surviving for more than 12 and 14 years with good lung functions.
Live related donor lobar lung transplantation; Live donor lobar lung transplantation; Lung transplantation
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 < 0.0001) and more organised sarcomere. The Ca2+ cycling properties of iPSC-CM cultured on these substrates were significantly altered with a shorter time to peak amplitude (p = 0.0002 at 1 Hz), and more organised sarcoplasmic reticulum (SR) Ca2+ release in response to caffeine (p < 0.0001), suggesting improved SR Ca2+ cycling. These changes were not associated with modifications in gene expression. Whilst structured tissue culture may make iPSC-CM more representative of adult myocardium, further construct development and characterisation is required to optimise iPSC-CM as a model of adult myocardium.
Calcium cycling; Cardiac tissue engineering; Electrophysiology; Micropatterning; Polydimethylsiloxane; Stem cells
The present study comprised sarcomeric genotyping of the three most commonly involved sarcomeric genes: MYBPC3, MYH7, and TNNT2 in 192 unrelated Egyptian hypertrophic cardiomyopathy (HCM) index patients. Mutations were detected in 40 % of cases. Presence of positive family history was significantly (p = 0.002) associated with a higher genetic positive yield (49/78, 62.8 %). The majority of the detected mutations in the three sarcomeric genes were novel (40/62, 65 %) and mostly private (47/62, 77 %). Single nucleotide substitution was the most frequently detected mutation type (51/62, 82 %). Over three quarters of these substitutions (21/27, 78 %) involved CpG dinucleotide sites and resulted from C > T or G > A transition in the three analyzed genes, highlighting the significance of CpG high mutability within the sarcomeric genes examined. This study could aid in global comparative studies in different ethnic populations and constitutes an important step in the evolution of the integrated clinical, translational, and basic science HCM program.
Sarcomeric genotyping; HCM genetics; Egypt
The transverse tubules (t-tubules) are invaginations of the cell membrane rich in several ion channels and other proteins devoted to the critical task of excitation–contraction coupling in cardiac muscle cells (cardiomyocytes). They are thought to promote the synchronous activation of the whole depth of the cell despite the fact that the signal to contract is relayed across the external membrane. However, recent work has shown that t-tubule structure and function are complex and tightly regulated in healthy cardiomyocytes. In this review, we outline the rapidly accumulating knowledge of its novel roles and discuss the emerging evidence of t-tubule dysfunction in cardiac disease, especially heart failure. Controversy surrounds the t-tubules' regulatory elements, and we draw attention to work that is defining these elements from the genetic and the physiological levels. More generally, this field illustrates the challenges in the dissection of the complex relationship between cellular structure and function.
t-tubule; heart failure; structure–function; cell membrane
Ca2+-induced Ca2+ release (CICR) is critical for contraction in cardiomyocytes. The transverse (t)-tubule system guarantees the proximity of the triggers for Ca2+ release [L-type Ca2+ channel, dihydropyridine receptors (DHPRs)] and the sarcoplasmic reticulum Ca2+ release channels [ryanodine receptors (RyRs)]. Transverse tubule disruption occurs early in heart failure (HF). Clinical studies of left ventricular assist devices in HF indicate that mechanical unloading induces reverse remodelling. We hypothesize that unloading of failing hearts normalizes t-tubule structure and improves CICR.
Methods and results
Heart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham-operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF-UN). HF reduced the t-tubule density measured by di-8-ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t-tubule system in HF was also reversed in HF-UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF-UN. Electron microscopy revealed recovery of normal t-tubule microarchitecture in HF-UN. L-type Ca2+ current density, measured using whole-cell patch clamping, was reduced in HF but unaffected by unloading. The variance of the time-to-peak of the Ca2+ transient, an index of CICR dyssynchrony, was increased in HF and normalized by unloading. The increased Ca2+ spark frequency observed in HF was reduced in HF-UN. These results could be explained by the recoupling of orphaned RyRs in HF, as indicated by immunofluorescence.
Our data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t-tubule system and improves CICR.
HF; Left ventricular assist device; Recovery
Prolonged mechanical unloading (UN) of the heart is associated with detrimental changes to the structure and function of cardiomyocytes. The mechanisms underlying these changes are unknown. In this study, we report the influence of UN on excitation-contraction coupling, Ca2+-induced Ca2+ release (CICR) in particular, and transverse (t)-tubule structure. UN was induced in male Lewis rat hearts by heterotopic abdominal heart transplantation. Left ventricular cardiomyocytes were isolated from the transplanted hearts after 4 wk and studied using whole-cell patch clamping, confocal microscopy, and scanning ion conductance microscopy (SICM). Recipient hearts were used as control (C). UN reduced the volume of cardiomyocytes by 56.5% compared with C (UN, n=90; C, n=59; P<0.001). The variance of time-to-peak of the Ca2+ transients was significantly increased in unloaded cardiomyocytes (UN 227.4±24.9 ms2, n=42 vs. C 157.8±18.0 ms2, n=40; P<0.05). UN did not alter the action potential morphology or whole-cell L-type Ca2+ current compared with C, but caused a significantly higher Ca2+ spark frequency (UN 3.718±0.85 events/100 μm/s, n=47 vs. C 0.908±0.186 events/100 μm/s, n=45; P<0.05). Confocal studies showed irregular distribution of the t tubules (power of the normal t-tubule frequency: UN 8.13±1.12×105, n=57 vs. C 20.60± 3.174×105, n=56; P<0.001) and SICM studies revealed a profound disruption to the openings of the t tubules and the cell surface in unloaded cardiomyocytes. We show that UN leads to a functional uncoupling of the CICR process and identify disruption of the t-tubule-sarcoplasmic reticulum interaction as a possible mechanism.—Ibrahim, M., Al Masri, A., Navaratnarajah, M., Siedlecka, U., Soppa, G. K., Moshkov, A., Abou Al-Saud, S., Gorelik, J., Yacoub, M. H., Terracciano, C. M. N. Prolonged mechanical unloading affects cardiomyocyte excitation-contraction coupling, transverse-tubule structure, and the cell surface.
Ca2+-induced Ca2+ release; scanning ion conductance microscope; confocal microscope
Endomyocardial Fibrosis (EMF) is a tropical restrictive cardiomyopathy of unknown etiology with high prevalence in Sub-Saharan Africa, for which it is unclear whether the primary target of injury is the endocardial endothelium, the subendocardial fibroblast, the coronary microcirculation or the myocyte. In an attempt to explore the possibility of endocardial lesions being a result of an immune response against the myocyte we assessed the presence and frequency of circulating anti-myocardial antibodies in EMF patients.
EMF classification, assessment of severity and staging was based on echocardiography. We used sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of myocardial proteins followed by western blotting to screen serum samples for antiheart antibodies G and M classes. The degree of serum reactivity was correlated with the severity and activity of EMF. We studied 56 EMF patients and 10 healthy controls. IgG reactivity against myocardial proteins was stronger and more frequent in patients with EMF when compared to controls (30/56; 53.6% vs. 1/10; 10%, respectively). IgM reactivity was weak in both groups, although higher in EMF patients (11/56; 19.6%) when compared to controls (n = 0). EMF patients showed greater frequency and reactivity of IgG antibodies against myocardial proteins of molecular weights 35 kD, 42 kD and 70 kD (p values <0.01, <0.01 and <0.05 respectively).
The presence of antibodies against myocardial proteins was demonstrated in a subset of EMF patients. These immune markers seem to be related with activity and might provide an adjunct tool for diagnosis and classification of EMF, therefore improving its management by identifying patients who may benefit from immunosuppressive therapy. Further research is needed to clarify the role of autoimmunity in the pathogenesis of EMF.
Endomyocardial Fibrosis is a tropical disease in which the heart cannot open properly to receive blood due to a scar that covers its inner layer. It affects mainly children and adolescents, and has a poor prognosis because the cause and mechanisms of scarring are unknown. The conventional treatment is frustrating and does not alter the natural history of the disease. Despite affecting several million people worldwide there has been little investigation on the mechanisms of the disease or drug development to improve its prognosis. In this study we investigate the presence of antibodies against the myocardial cells of African patients with severe and advanced EMF aiming at uncovering new pathways for the disease. Our results reveal that EMF patients have anti-myocardial antibodies in their blood. The reaction of these antibodies with the heart may be one of the mechanisms involved in the genesis of the fibrotic lesions. This knowledge may help in diagnosing the condition and provide alternatives for its management, using drugs that reduce the impact of the circulating antibodies in the cardiac tissue. The significance of these results needs confirmation on studies involving larger number of subjects due to frequent finding of antiheart antibodies in African populations with heart failure of any cause.
Elastin is a vital structural and regulatory matrix protein that plays an important role in conferring elasticity to blood vessel wall. Previous tissue engineering approaches to regenerate elastin in situ or within tissue engineering constructs are curtailed by innate poor elastin synthesis potential by adult vascular smooth muscle cells (SMCs). Currently, we seek to develop cellular cues to enhance tropoelastin synthesis and improve elastin matrix yield, stability, and ultrastructure. Our earlier studies attest to the elastogenic utility of hyaluronan (HA)–based cellular cues, though their effects are fragment size dependent and dose dependent, with HA oligomers deemed most elastogenic. We presently show transforming growth factor beta 1 (TGF-β1) and HA oligomers, when provided concurrently, to synergistically and dramatically improve elastin matrix regeneration by adult vascular SMCs. Together, these cues suppress SMC proliferation, enhance synthesis of tropoelastin (8-fold) and matrix elastin protein (5.5-fold), and also improve matrix elastin yield (45% of total elastin vs. 10% for nonadditive controls), possibly by more efficient recruitment of tropoelastin for crosslinking. The density of desmosine crosslinks within the elastin matrix was itself attenuated, although the cues together modestly increased production and activity of the elastin crosslinking enzyme, lysyl oxidase. TGF-β1 and HA oligomers together induced much greater assembly of mature elastin fibers than they did separately, and did not induce matrix calcification. The present outcomes might be great utility to therapeutic regeneration of elastin matrix networks in situ within elastin-compromised vessels, and within tissue-engineered vascular graft replacements.
Although recent progress in cardiovascular tissue engineering has generated great expectations for the exploitation of stem cells to restore cardiac form and function, the prospects of a common mass-produced cell resource for clinically viable engineered tissues and organs remain problematic. The refinement of stem cell culture protocols to increase induction of the cardiomyocyte phenotype and the assembly of transplantable vascularized tissue are areas of intense current research, but the problem of immune rejection of heterologous cell type poses perhaps the most significant hurdle to overcome. This article focuses on the potential advantages and problems encountered with various stem cell sources for reconstruction of the damaged or failing myocardium or heart valves and also discusses the need for integrating advances in developmental and stem cell biology, immunology and tissue engineering to achieve the full potential of cardiac tissue engineering. The ultimate goal is to produce ‘off-the-shelf’ cells and tissues capable of inducing specific immune tolerance.
stem cells; tolerance; immune response; regenerative medicine; tissue engineering
Intramyocardial injection of skeletal myoblasts (SMB) has been shown to be a promising strategy for treating post-infarction chronic heart failure. However, insufficient therapeutic benefit and occurrence of ventricular arrhythmias are concerns. We hypothesised that the use of a retrograde intracoronary route for SMB-delivery might favourably alter the behaviour of the grafted SMB, consequently modulating the therapeutic effects and arrhythmogenicity.
Methods and Results
Three weeks after coronary artery ligation in female wild-type rats, 5×106 GFP-expressing SMB or PBS only (control) were injected via either the intramyocardial or retrograde intracoronary routes. Injection of SMB via either route similarly improved cardiac performance and physical activity, associated with reduced cardiomyocyte-hypertrophy and fibrosis. Grafted SMB via either route were only present in low numbers in the myocardium, analysed by real-time PCR for the Y-chromosome specific gene, Sry. Cardiomyogenic differentiation of grafted SMB was extremely rare. Continuous ECG monitoring by telemetry revealed that only intramyocardial injection of SMB produced spontaneous ventricular tachycardia up to 14 days, associated with local myocardial heterogeneity generated by clusters of injected SMB and accumulated inflammatory cells. A small number of ventricular premature contractions with latent ventricular tachycardia were detected in the late-phase of SMB injection regardless of the injection-route.
Retrograde intracoronary injection of SMB provided significant therapeutic benefits with attenuated early-phase arrhythmogenicity in treating ischaemic cardiomyopathy, indicating the promising utility of this route for SMB-delivery. Late-phase arrhythmogenicity remains a concern, regardless of the delivery route.
To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH).
Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.
We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.
Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.
Heterotopic tracheal allografts in small rodents have been shown to share many characteristics with the development of obliterative bronchiolitis (OB) in the clinic and therefore provide a suitable animal model for the study of OB. The model facilitates the examination of the pathogenesis of the disease and the elucidation of the cellular and molecular mechanisms involved in its development. The model provides a less technically demanding alternative to whole lung transplantation in small rodents and should lead to a speedier identification of new treatments that might prevent the development of post-transplantation OB in the clinic.
allograft; model; obliterative bronchiolitis; rat; trachea
A murmur of mitral regurgitation in patients with congestive heart failure after an acute myocardial infarction suggests a surgically correctable cause of the heart failure. Six patients who presented in this manner and who later underwent surgery have been evaluated by two-dimensional echocardiography (2DE). The underlying anatomical cause of the mitral regurgitation was correctly identified as papillary muscle rupture (2 cases), ruptured chordae tendineae (1 case) and papillary muscle fibrosis (3 cases).
Two-dimensional echocardiography is useful in evaluating patients with congestive heart failure who develop a systolic murmur after acute infarction, as it can detect surgically correctable structural defects.
Idiopathic pulmonary hilar fibrosis is a condition related to mediastinal fibrosis, characterized by localization of the fibrosing process to one or both pulmonary hila. This results in pulmonary hypertension and bronchial narrowing. Three patients suffering from this disease, in whom the diagnosis has been confirmed by thoracotomy, are reported. The clinical and pathological features are described and previously reported cases are reviewed. The syndrome is classified into two types, according to whether the obstruction affects mainly the pulmonary artery or veins. The disease is a self-limiting one but may lead to organic changes in the lungs causing severe disability.
Valve interstitial cells populate aortic valve cusps and have been implicated in aortic valve calcification. Here we investigate a common in vitro model for aortic valve calcification by characterizing nodule formation in porcine aortic valve interstitial cells (PAVICs) cultured in osteogenic (OST) medium supplemented with transforming growth factor beta 1 (TGF-β1). Using a combination of materials science and biological techniques, we investigate the relevance of PAVICs nodules in modeling the mineralised material produced in calcified aortic valve disease. PAVICs were grown in OST medium supplemented with TGF-β1 (OST+TGF-β1) or basal (CTL) medium for up to 21 days. Murine calvarial osteoblasts (MOBs) were grown in OST medium for 28 days as a known mineralizing model for comparison. PAVICs grown in OST+TGF-β1 produced nodular structures staining positive for calcium content; however, micro-Raman spectroscopy allowed live, noninvasive imaging that showed an absence of mineralized material, which was readily identified in nodules formed by MOBs and has been identified in human valves. Gene expression analysis, immunostaining, and transmission electron microscopy imaging revealed that PAVICs grown in OST+TGF-β1 medium produced abundant extracellular matrix via the upregulation of the gene for Type I Collagen. PAVICs, nevertheless, did not appear to further transdifferentiate to osteoblasts. Our results demonstrate that ‘calcified’ nodules formed from PAVICs grown in OST+TGF-β1 medium do not mineralize after 21 days in culture, but rather they express a myofibroblast-like phenotype and produce a collagen-rich extracellular matrix. This study clarifies further the role of PAVICs as a model of calcification of the human aortic valve.