W. Andrew Owens

Telomerase expression in the mammalian heart

While the mammalian heart has low, but functionally significant, levels of telomerase expression, the cellular population responsible remains incompletely characterized. This study aimed to identify the cell types responsible for cardiac telomerase activity in neonatal, adult, and cryoinjured adult hearts using transgenic mice expressing green fluorescent protein (GFP), driven by the promoter for murine telomerase reverse transcriptase (mTert), which is a necessary and rate‐limiting component of telomerase. A rare population of mTert‐GFP‐expressing cells was identified that possessed all detectable cardiac telomerase RNA and telomerase activity. It was heterogeneous and included cells coexpressing markers of cardiomyocytic, endothelial, and mesenchymal lineages, putative cardiac stem cell markers, and, interestingly, cardiomyocytes with a differentiated phenotype. Quantification using both flow cytometry and immunofluorescence identified a significant decline in mTert‐GFP cells in adult animals compared to neonates (~9‐ and ~20‐fold, respectively). Cardiac injury resulted in a ~6.45‐fold expansion of this population (P<0.005) compared with sham‐operated controls. This study identifies the cells responsible for cardiac telomerase activity, demonstrates a significant diminution with age but a marked response to injury, and, given the relationship between telomerase activity and stem cell populations, suggests that they represent a potential target for further investigation of cardiac regenerative potential.—Richardson, G. D., Breault, D., Horrocks, G., Cormack, S., Hole, N., Owens, W. A. Telomerase expression in the mammalian heart. FASEB J. 26, 4832–4840 (2012). www.fasebj.org

Genetic variants associated with risk of atrial fibrillation regulate expression of PITX2, CAV1, MYOZ1, C9orf3 and FANCC.

Genome-wide association studies (GWAS) have identified genetic variants in a number of chromosomal regions that are associated with atrial fibrillation (AF). The mechanisms underlying these associations are unknown, but are likely to involve effects of the risk haplotypes on expression of neighbouring genes. To investigate the association between genetic variants at AF-associated loci and expression of nearby candidate genes in human atrial tissue and peripheral blood. Right atrial appendage (RAA) samples were collected from 122 patients undergoing cardiac surgery, of these, 12 patients also had left atrial appendage samples taken. 22 patients had a history of AF. Peripheral blood samples were collected from 405 patients undergoing diagnostic cardiac catheterisation. In order to tag genetic variation at each of nine loci, a total of 367 single nucleotide polymorphisms (SNPs) were genotyped using the Sequenom platform. Total expression of 16 candidate genes in the nine AF-associated regions was measured by quantitative PCR. The relative expression of each allele of the candidate genes was measured on the Sequenom platform using one or more transcribed SNPs to distinguish between alleles in heterozygotes. We tested association between the SNPs of interest and gene expression using total gene expression (integrating cis and trans acting sources of variation), and allelic expression ratios (specific for cis acting influences), in atrial tissue and peripheral blood. We adjusted for multiple comparisons using a Bonferroni approach. In subsidiary analyses, we compared the expression of candidate genes between patients with and without a history of AF. Total expression of 15 transcripts of 14 genes and allelic expression ratio of 14 transcripts of 14 genes in genomic regions associated with AF were measured in right atrial appendage tissue. 8 of these transcripts were also expressed in peripheral blood. Risk alleles at AF-associated SNPs were associated in cis with an increased expression of PITX2a (2.01-fold, p=6.5×10(-4)); and with decreased expression of MYOZ1 (0.39 fold; p=5.5×10(-15)), CAV1 (0.89 fold; p=5.9×10(-8)), C9orf3 (0.91 fold; 1.5×10(-5)), and FANCC (0.94-fold; p=8.9×10(-8)) in right atrial appendage. Of these five genes, only CAV1 was expressed in peripheral blood; association between the same AF risk alleles and lower expression of CAV1 was confirmed (0.91 fold decrease; p=4.2×10(-5)). A history of AF was also associated with a decrease in expression of CAV1 in both right and left atria (0.84 and 0.85 fold, respectively; p=0.03), congruent with the magnitude of the effect of the risk SNP on expression, and independent of genotype. The analyses in peripheral blood showed association between AF risk SNPs and decreased expression of KCNN3 (0.85-fold; p=2.1×10(-4)); and increased expression of SYNE2 (1.12-fold; p=7.5×10(-24)); however, these associations were not detectable in atrial tissue. We identified novel cis-acting associations in atrial tissue between AF risk SNPs and increased expression of PITX2a/b; and decreased expression of CAV1 (an association also seen in peripheral blood), C9orf3 and FANCC. We also confirmed a previously described association between AF risk variants and MYOZ1 expression. Analyses of peripheral blood illustrated tissue-specificity of cardiac eQTLs and highlight the need for larger-scale genome-wide eQTL studies in cardiac tissue. Our results suggest novel aetiological roles for genes in four AF-associated genomic regions.

Late Results of Patch Repair of Coarctation of the Aorta in Adults Using Autogenous Arterial Wall

BACKGROUND Interposition grafting or patch repair of adult coarctations of the aorta are the standard methods of surgical treatment. Both involve use of prosthetic material, and patch repair using prosthetic material may lead to aneurysm formation in the long term. METHODS Four patients aged 17 to 29 years had been investigated for systemic hypertension and had coarctation of the aorta diagnosed on cardiac catheterization. Between March and November 1984, all 4 underwent a corrective operation. The lesions were widely incised and a broad patch of ipsilateral mammary or Abbotts artery was fashioned across the narrowing. The arteries had been enlarged in diameter because of prolonged exposure to high blood pressure as collateral vessels, although none was intrinsically diseased. RESULTS After 12 years of follow-up, only 1 patient remains on antihypertensive therapy. Spiral computed tomographic reconstructions revealed only very mild residual stenosis in 1 patient, confirmed by subsequent aortography. CONCLUSIONS In adult patients with coarctation of the aorta, the use of the enlarged internal mammary artery as a patch graft is a simple, quick procedure, which may give lasting relief of obstruction. Spiral computed tomographic scanning is an ideal noninvasive method of follow-up.

Secondary prevention following coronary artery bypass grafting has improved but remains sub-optimal: the need for targeted follow-up

A focused review of secondary preventive medication following revascularisation provides an opportunity to ensure optimal use of these agents. A retrospective analysis of our in-house cardiothoracic surgical database was performed to identify patients undergoing non-emergency, elective surgical revascularisation discharged on four secondary preventive medications: aspirin; beta-blockers; ACE-inhibitors and statins. Of 2749 patients studied, 2302 underwent isolated coronary artery bypass grafting (CABG), mean age 65.5 years (S.D. 9.15). Overall, 2536 (92%) patients were prescribed aspirin. Beta-blockers were prescribed in 2171 (79%) patients overall, in 1096/1360 (81%) of patients with a history of myocardial infarction and in 465/619 (75%) of patients with left ventricular systolic dysfunction (LVSD). Overall, 1518 (55%) patients were prescribed an ACE-inhibitor and 179 (6.5%) an angiotensin receptor blocker (ARB); one of these agents was prescribed in 446/619 (72%) patients with LVSD and 915/1360 (67%) patients with a history of previous myocardial infarction. Overall, 2518 (92%) patients were prescribed a statin. Secondary preventive therapies are prescribed more commonly on discharge after CABG than in previous studies, but there is a continuing under-utilisation of ACE-inhibitors. To maximise the potential benefits of these agents, further study is required to understand why they are not prescribed.

Coronary bypass grafting using crossclamp fibrillation does not result in reliable reperfusion of the myocardium when the crossclamp is intermittently released: a prospective cohort study

BackgroundCross-clamp fibrillation is a well established method of performing coronary grafting, but its clinical effect on the myocardium is unknown. We sought to measure these effects clinically using the Khuri Intramyocardial pH monitor.Methods50 episodes of cross-clamping were recorded in 16 patients who underwent CABG with crossclamp-fibrillation. An Intramyocardial pH probe measured the level of acidosis in the anterior and posterior myocardium in real-time. The pH at the start and end of each period of cross-clamping was recorded.ResultsIt became very apparent that the pH of some patients recovered quickly while others entirely failed to recover. Thus the patients were split into 2 groups according to whether the pH recovered to above 6.8 after the first crossclamp-release (N = 8 in each group). Initial pH was 7.133 (range 6.974–7.239). After the first period of crossclamping the pH dropped to 6.381 (range 6.034–6.684). The pH in recoverers prior to the second XC application was 6.990(range 6.808–7.222) compared to only 6.455 (range 6.200–6.737) in patients whose myocardium did not recover (P < 0.0005). This finding was repeated after the second XC release (mean pH 7.005 vs 6.537) and the third (mean pH 6.736 vs 6.376). However prior to separation from bypass the pH was close to the initial pH in both groups (7.062 vs 7.038).ConclusionCrossclamp fibrillation does not result in reliable reperfusion of the myocardium between periods of crossclamping.

A policy of elective delayed sternal closure does not improve the outcome after arterial switch.

BACKGROUND Delayed sternal closure is regularly used in the immediate management of hemodynamic instability after neonatal cardiac procedures. The aim of this study was to assess whether the routine, elective use of delayed sternal closure would reduce morbidity in neonates undergoing arterial switch for transposition of the great arteries. METHODS A retrospective statistical analysis was performed on 52 neonates operated on from 1991 to 1998. Until 1994, chest closure was routinely attempted in all patients after arterial switch; the policy was then changed to delayed sternal closure in all cases in the latter half of the study period. RESULTS Delayed sternal closure did not significantly alter the mean duration of ventilation (2.7 +/- 2.37 versus 2.7 +/- 1.3 days) nor intensive care stay (4.1 +/- 2.8 versus 5.7 +/- 10.0 days; p = 0.46). There was no increase in the incidence of wound sepsis (7.7% versus 3.8%; p = 0.55), and mortality was unchanged (7.7% in both groups). There was an increase in the incidence of urgent reexploration (7.7% versus 19.2%; p = 0.22), which did not reach significance. CONCLUSIONS This study does not support the hypothesis that elective delayed sternal closure reduces the morbidity after arterial switch in neonates but does, however, confirm the safety and efficacy of the procedure.

Video-assisted thoracoscopic left ventricular pacing in patients with and without previous sternotomy.

BACKGROUND Left ventricular epicardial lead placement via video-assisted thoracoscopy (VAT) is a recognized surgical technique to achieve cardiac resynchronization therapy (CRT) when conventional lead placement has failed. Its role in patients with previous sternotomy is uncertain. We describe our experience in a cohort of patients including those with previous sternotomy. METHODS This was a retrospective review of consecutive patients undergoing VAT lead implantation for CRT in a single center between 2004 and 2011. All patients fulfilled conventional criteria for CRT and were followed up at 4 to 6 weeks and then at 3-month intervals. Clinical and pacing parameters were compared at baseline and at the latest review. RESULTS Thirty-two patients (27 men; mean age, 67 ± 9 years) underwent VAT left ventricular lead implantation. Mean follow-up duration was 704 ± 450 days. Ten patients (31%) had undergone previous sternotomy. Thoracoscopic lead implantation was successful in 31 patients (97%): 1 patient with two previous sternotomies required conversion to open thoracotomy due to bleeding with multiple adhesions. Satisfactory implantation pacing thresholds of 2 volts or less at 0.5 ms were achieved in all patients. Despite a longer operative time in those with previous sternotomy, all clinical and pacing outcomes, including complications, clinical response to CRT, and long-term pacing variables were similar between the groups. CONCLUSIONS VAT left ventricular lead placement appears safe and effective in selected patients with previous sternotomy, including coronary artery bypass operations, with postoperative outcomes comparable with those patients without previous sternotomy.

Manubrium-limited sternotomy decreases blood loss after aortic valve replacement surgery

OBJECTIVES Minimally invasive surgical approaches for aortic valve replacement (AVR) are growing in popularity in an attempt to decrease morbidity from conventional surgery. We have adopted a technique that divides only the manubrium and spares the body of the sternum. We sought to determine whether patients benefit from this less-invasive approach. METHODS We retrospectively analysed our prospectively maintained database to review all isolated aortic valve replacements performed in an 18-month period from November 2011 to April 2013. RESULTS One hundred and ninety-one patients were identified, 98 underwent manubrium-limited sternotomy (Mini-AVR) and 93 had a conventional median sternotomy (AVR). The two groups were well matched for preoperative variables and risk (mean logistic EuroSCORE mini-AVR 7.15 vs AVR 6.55, P = 0.47). Mean cardiopulmonary bypass and aortic cross-clamp times were 10 and 6 min longer, respectively, in the mini-AVR group (mean values 88 vs 78 min, P = 0.00040, and 66 vs 60 min, P = 0.0078, respectively). Mini-AVR patients had significantly less postoperative blood loss, 332 vs 513 ml, P = 0.00021, and were less likely to require blood products (fresh-frozen plasma and platelets), 24 vs 36%, P = 0.042. Postoperative complications and length of stay were similar (discharge on or before Day 4; mini-AVR 15 vs AVR 8%, P = 0.17). Valve outcome (paravalvular leak mini-AVR 2 vs AVR 1%, P = 1.00) and survival (mini-AVR 99 vs AVR 97%, P = 0.36) were equal. CONCLUSIONS A manubrium-limited approach maintains outcomes achieved for aortic valve replacement by conventional sternotomy while significantly reducing postoperative blood loss and transfusion of blood products.

Optimised Protocols for the Identification of the Murine Cardiac Side Population

Cardiac side population (CSP) cells, defined by their ability to efflux the vital dye Hoechst 33342, have been identified as putative cardiac stem cells based on their potential to give rise to both cardiomyocytes and endothelial cells. The CSP phenotype relies on an active metabolic pathway and cell viability to identify a rare population of cells and therefore technical differences in the CSP staining protocol can lead to inconsistent results and discrepancies between studies. Here we describe an established protocol for CSP identification and have optimised a protocol for CSP analysis utilizing an automated cardiac digestion procedure using gentleMACs dissociation and Hoechst 33342 staining followed by dual wavelength flow cytometric analysis.

Dermal stem cells can differentiate down an endothelial lineage.

In this study, we have demonstrated that cells of neural crest origin located in the dermal papilla (DP) exhibit endothelial marker expression and a functional activity. When grown in endothelial growth media, DP primary cultures upregulate expression of vascular endothelial growth factor receptor 1 (FLT1) mRNA and downregulate expression of the dermal stem cell marker α-smooth muscle actin. DP cells have demonstrated functional characteristics of endothelial cells, including the ability to form capillary-like structures on Matrigel, increase uptake of low-density lipoprotein and upregulate ICAM1 (CD54) in response to tumour necrosis factor alpha (TNF-α) stimulation. We confirmed that these observations were not due to contaminating endothelial cells, by using DP clones. We have also used the WNT1cre/ROSA26R and WNT1cre/YFP lineage-tracing mouse models to identify a population of neural crest-derived cells in DP cultures that express the endothelial marker PECAM (CD31); these cells also form capillary-like structures on Matrigel. Importantly, cells of neural crest origin that express markers of endothelial and mesenchymal lineages exist within the dermal sheath of the vibrissae follicle.