Proteome analysis in cardiovascular pathophysiology using Dahl rat model

Dahl salt-sensitive (DS) and salt-resistant (DR) inbred rat strains represent a well established animal model for cardiovascular research. Upon prolonged administration of high-salt-containing diet, DS rats develop systemic hypertension, and as a consequence they develop left ventricular hypertrophy, followed by heart failure. The aim of this work was to explore whether this animal model is suitable to identify biomarkers that characterize defined stages of cardiac pathophysiological conditions. The work had to be performed in two stages: in the first part proteomic differences that are attributable to the two separate rat lines (DS and DR) had to be established, and in the second part the process of development of heart failure due to feeding the rats with high-salt-containing diet has to be monitored. This work describes the results of the first stage, with the outcome of protein expression profiles of left ventricular tissues of DS and DR rats kept under low salt diet. Substantial extent of quantitative and qualitative expression differences between both strains of Dahl rats in heart tissue was detected. Using Principal Component Analysis, Linear Discriminant Analysis and other statistical means we have established sets of differentially expressed proteins, candidates for further molecular analysis of the heart failure mechanisms.

Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish.

The zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is based on the removal of heart tissue rather than on tissue damage. We recently characterized the cellular response and regenerative capacity of the zebrafish heart after cryoinjury (CI), an alternative procedure that more closely models the pathophysiological process undergone by the human heart after myocardial infarction (MI). After anesthesia, localized CI with a liquid nitrogen-cooled copper probe induced damage in 25% of the ventricle, in a procedure requiring <5 min. Here we present a detailed description of the technique, which provides a valuable system for the study of the mechanisms of heart regeneration and scar removal after MI in a versatile vertebrate model.

Transcriptional response to cardiac injury in the zebrafish: systematic identification of genes with highly concordant activity across in vivo models

BACKGROUND Zebrafish is a clinically-relevant model of heart regeneration. Unlike mammals, it has a remarkable heart repair capacity after injury, and promises novel translational applications. Amputation and cryoinjury models are key research tools for understanding injury response and regeneration in vivo. An understanding of the transcriptional responses following injury is needed to identify key players of heart tissue repair, as well as potential targets for boosting this property in humans. RESULTS We investigated amputation and cryoinjury in vivo models of heart damage in the zebrafish through unbiased, integrative analyses of independent molecular datasets. To detect genes with potential biological roles, we derived computational prediction models with microarray data from heart amputation experiments. We focused on a top-ranked set of genes highly activated in the early post-injury stage, whose activity was further verified in independent microarray datasets. Next, we performed independent validations of expression responses with qPCR in a cryoinjury model. Across in vivo models, the top candidates showed highly concordant responses at 1 and 3 days post-injury, which highlights the predictive power of our analysis strategies and the possible biological relevance of these genes. Top candidates are significantly involved in cell fate specification and differentiation, and include heart failure markers such as periostin, as well as potential new targets for heart regeneration. For example, ptgis and ca2 were overexpressed, while usp2a, a regulator of the p53 pathway, was down-regulated in our in vivo models. Interestingly, a high activity of ptgis and ca2 has been previously observed in failing hearts from rats and humans. CONCLUSIONS We identified genes with potential critical roles in the response to cardiac damage in the zebrafish. Their transcriptional activities are reproducible in different in vivo models of cardiac injury.

Cell therapies for heart function recovery: focus on myocardial tissue engineering and nanotechnologies

Cell therapies have gained increasing interest and developed in several approaches related to the treatment of damaged myocardium. The results of multiple clinical trials have already been reported, almost exclusively involving the direct injection of stem cells. It has, however, been postulated that the efficiency of injected cells could possibly be hindered by the mechanical trauma due to the injection and their low survival in the hostile environment. It has indeed been demonstrated that cell mortality due to the injection approaches 90%. Major issues still need to be resolved and bed-to-bench followup is paramount to foster clinical implementations. The tissue engineering approach thus constitutes an attractive alternative since it provides the opportunity to deliver a large number of cells that are already organized in an extracellular matrix. Recent laboratory reports confirmed the interest of this approach and already encouraged a few groups to investigate it in clinical studies. We discuss current knowledge regarding engineered tissue for myocardial repair or replacement and in particular the recent implementation of nanotechnological approaches.

Relationship between heart rate variability, interleukin-6, and soluble tissue factor in healthy subjects

Decreased heart rate variability (HRV) has been associated with an increased risk of atherosclerosis. We hypothesized that a decrease in frequency domains of resting HRV would be associated with elevated plasma levels of interleukin (IL)-6 and soluble tissue factor (sTF) both previously shown to prospectively predict atherothrombotic events in healthy subjects. Subjects were 102 healthy and unmedicated black and white middle-aged men and women. We determined IL-6 and sTF antigen in plasma and HRV measures from surface electrocardiogram data using spectral analysis. All statistical analyses controlled for age, gender, ethnicity, smoking status, blood pressure, and body mass index. Low amounts of low frequency (LF) power (beta=-0.31, p=0.007) and high frequency (HF) power (beta=-0.36, p=0.002) were associated with increased amounts of IL-6, explaining 7% and 9% of the variance, respectively. Interactions between LF power and IL-6 (p=0.002) and between HF power and IL-6 (p=0.012) explained 8% and 5%, respectively, of the variance in sTF. Post hoc analyses showed associations between IL-6 and sTF when LF power (beta=0.51, p<0.001) and HF power (beta=0.48, p<0.001) were low but not when LF power and high HF power were high. The findings suggest that systemic low-grade inflammatory activity is associated with a decrease in HRV. Furthermore, there was a positive relationship between plasma levels of IL-6 and sTF antigen when HRV was low. Inflammation and related hypercoagulability might particularly contribute to atherothrombotic events in a setting of decreased HRV.

Comparison between pulsed-wave Doppler- and tissue Doppler-derived Tei indices in fetuses with and without congenital heart disease

OBJECTIVES The aim of this study was to compare the right (RV) and left (LV) ventricular Tei indices obtained by pulsed-wave Doppler (PD) and tissue Doppler (TD) methods in fetuses with structurally normal and abnormal hearts. METHODS This was a retrospective cross-sectional study of 147 fetuses that had a fetal echocardiogram and Tei index measured during a 2-year period. The RV and LV Tei indices were measured using both PD and TD methods. The difference between the two methods of Tei index measurement was tested using paired sample t-test, Pearson correlation coefficient was used to examine their relationship, and the agreement between the methods was tested using Bland-Altman analysis. RESULTS A total of 87 fetuses had normal hearts and 60 had a congenital heart defect. Both PD and TD Tei indices were measured successfully from at least one ventricle in 123 cases and from both ventricles in 110 cases. The mean TD Tei index was significantly higher than the mean PD Tei index for both ventricles (P < 0.0001). There was a weak but statistically significant correlation between the PD and TD Tei indices of the right ventricle (r = 0.20, P = 0.029), whereas the PD and TD Tei indices of the left ventricle did not correlate significantly (r = 0.04, P = 0.684). When pairs of Tei indices measured by two different methods (123 pairs for the right ventricle and 111 for the left ventricle) were tested with Bland-Altman analysis, the bias and precision were 0.147 and 0.254, respectively, for the right ventricle, and 0.299 and 0.276, respectively, for the left ventricle. CONCLUSIONS Correlation between Tei indices measured by PD and TD methods is weak and the agreement between individual measurements is poor. Therefore, they should not be used interchangeably in the assessment of fetal cardiac function.

BPAG1 isoform-b: Complex distribution pattern in striated and heart muscle and association with plectin and α-actinin

BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein alpha-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like alpha-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and alpha-actinin-2, and its NH(2)-terminus, which contains an actin-binding domain, directly interacted with that of plectin and alpha-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.

Assessment of right ventricular systolic function: Comparison between cardiac magnetic resonance derived ejection fraction and pulsed-wave tissue Doppler imaging of the tricuspid annulus

Systolic right ventricular (RV) function is an important predictor in the course of various congenital and acquired heart diseases. Its practical determination by echocardiography remains challenging. We compared routine assessment of lateral tricuspid annular systolic motion velocity (TV(lat), cm/s) using pulsed-wave tissue Doppler imaging from the apical 4-chamber view with cardiac magnetic resonance (CMR) as reference method.

Correlation of fat embolism severity and subcutaneous fatty tissue crushing and bone fractures

Pulmonary fat embolism (PFE) is frequently encountered in blunt trauma. The clinical manifestation ranges from no impairment in light cases to death due to right-sided heart failure or hypoxaemia in severe cases. Occasionally, pulmonary fat embolism can give rise to a fat embolism syndrome (FES), which is marked by multiorgan failure, respiratory disorders, petechiae and often death. It is well known that fractures of long bones can lead to PFE. Several authors have argued that PFE can arise due to mere soft tissue injury in the absence of fractures, a claim other authors disagree upon. In this study, we retrospectively examined 50 victims of blunt trauma with regard to grade and extent of fractures and crushing of subcutaneous fatty tissue and presence and severity of PFE. Our results indicate that PFE can arise due to mere crushing of subcutaneous fat and that the fracture grade correlated well with PFE severity (p = 0.011). The correlation between PFE and the fracture severity (body regions affected by fractures and fracture grade) showed a lesser significant correlation (p = 0.170). The survival time (p = 0.567), the amount of body regions affected by fat crushing (p = 0.336) and the fat crush grade (p = 0.485) did not correlate with the PFE grade, nor did the amount of body regions affected by fractures. These results may have clinical implications for the assessment of a possible FES development, as, if the risk of a PFE is known, preventive steps can be taken.

Pulsed-wave tissue Doppler echocardiography for the analysis of fetal cardiac arrhythmias

Rhythm analysis of the fetal heart is hampered by the inability to routinely obtain electrocardiographic recordings of the fetus. Doppler studies of fetal cardiac tissue movements, assessing cardiac movements both qualitatively and quantitatively, have recently been described. We used a conventional high-resolution ultrasound system to obtain rhythm data from pulsed-wave tissue Doppler signals of the fetal heart in normal cardiac rhythm and in a variety of fetal cardiac arrhythmias.

Long-term tissue coverage of a biodegradable polylactide polymer-coated biolimus-eluting stent: comparative sequential assessment with optical coherence tomography until complete resorption of the polymer

Biolimus-eluting stents (BESs) with a biodegradable polymer in abluminal coating achieve more complete coverage at 9 months compared with sirolimus-eluting stents (SESs) with a durable polymer, as assessed by optical coherence tomography (OCT). Whether this advantage persists or augments after complete resorption of the polymer (>12 months) is unknown.

Tissue coverage of a hydrophilic polymer-coated zotarolimus-eluting stent vs. a fluoropolymer-coated everolimus-eluting stent at 13-month follow-up: an optical coherence tomography substudy from the RESOLUTE All Comers trial

Aims To compare the tissue coverage of a hydrophilic polymer-coated zotarolimus-eluting stent (ZES) vs. a fluoropolymer-coated everolimus-eluting stent (EES) at 13 months, using optical coherence tomography (OCT) in an ‘all-comers' population of patients, in order to clarify the mechanism of eventual differences in the biocompatibility and thrombogenicity of the devices. Methods and results Patients randomized to angiographic follow-up in the RESOLUTE All Comers trial (NCT00617084) at pre-specified OCT sites underwent OCT follow-up at 13 months. Tissue coverage and apposition were assessed strut by strut, and the results in both treatment groups were compared using multilevel logistic or linear regression, as appropriate, with clustering at three different levels: patient, lesion, and stent. Fifty-eight patients (30 ZES and 28 EES), 72 lesions, 107 stents, and 23 197 struts were analysed. Eight hundred and eighty-seven and 654 uncovered struts (7.4 and 5.8%, P= 0.378), and 216 and 161 malapposed struts (1.8 and 1.4%, P= 0.569) were found in the ZES and EES groups, respectively. The mean thickness of coverage was 116 ± 99 µm in ZES and 142 ± 113 µm in EES (P= 0.466). No differences in per cent neointimal volume obstruction (12.5 ± 7.9 vs. 15.0 ± 10.7%) or other areas–volumetric parameters were found between ZES and EES, respectively. Conclusion No significant differences in tissue coverage, malapposition, or lumen/stent areas and volumes were detected by OCT between the hydrophilic polymer-coated ZES and the fluoropolymer-coated EES at 13-month follow-up.

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Cardiac tissue-restricted deletion of plakoglobin results in progressive cardiomyopathy and activation of {beta}-catenin signaling

Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/beta-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase beta-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3beta. Finally, beta-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects beta-catenin activity in the heart and its implications for disease pathogenesis.