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Human heart samples from the Sydney Heart Bank have become a de facto standard against which others can be measured. Crucially, the heart bank contains a lot of donor heart material: for most researchers this is the hardest to obtain and yet is necessary since we can only study the pathological human heart in comparison with a control, preferably a normal heart sample. It is not generally realised how important the control is for human heart studies. We review our studies on donor heart samples. We report the results obtained with 17 different donor samples collected from 1994 to 2011 and measured from 2005 to 2015 by our standard methodology for in vitro motility and troponin I phosphorylation measurements. The donor heart sample parameters are consistent between the hearts, over time and with different operators indicating that Sydney Heart Bank donor hearts are a valid baseline control for comparison with pathological heart samples. We also discuss to what extent donor heart samples are representative of the normal heart. Electronic supplementary material The online version of this article (10.1007/s12551-020-00740-2) contains supplementary material, which is available to authorized users.

Background Smoking and dyslipidaemia are known individual risk factors of coronary artery disease (CAD). The present study examined the combined risk of smoking and dyslipidaemia on coronary atherosclerosis. Methods Coronary artery calcium (CAC), measured by cardiac CT, was used to assess the extent of CAD, which was related to smoking and dyslipidaemia using logistic regression, adjusted for age, sex, hypertension, BMI and family history of ischaemic heart disease. Results Seventy-one patients (46 men, 25 women: median age of 53.7yrs; IQR = 47.0–59.5) were recruited. The mean log10 CAC score in never-smokers without dyslipidaemia (reference group) was 0.37 (SD = 0.73), while the value in those with a history of smoking was 0.44 ± 0.48 (mean difference: 0.07, 95%CI:–0.67 to 0.81, p = 0.844), dyslipidaemia was 1.07 ± 1.08 (mean difference: 0.71, 95%CI: 0.24 to 1.17, p = 0.003), and both risk factors was 1.82 ± 0.64 (mean difference: 1.45, 95%CI:0.88 to 2.02, p Conclusions Smoking and dyslipidaemia together are associated with high coronary artery calcification and CAD, independent of other major risk factors.

Background Left ventricular ejection fraction (LVEF) is generally measured by echocardiography but is increasingly available with myocardial perfusion scintigraphy. With myocardial perfusion scintigraphy, the threshold of LVEF below which there is a risk for myocardial infarct or sudden cardiac death is higher for women (51%) than for men (43%). We tested the hypothesis that such a sex difference may also occur with echocardiography and myocardial perfusion scintigraphy. Methods Four hundred and four men, mean age = 67.7 ± SD = 12.3 yr; 339 women, 67.7 ± 11.7 yr had separate myocardial perfusion scintigraphy and echocardiography examinations within six months. A subset of 327 of these patients (181 men, 68.8 ± 12.1 yr; 146 women, 66.4 ± 12.1 yr) had examinations within one month and were additionally analysed as this sub-group. Myocardial perfusion scintigraphy and echocardiography were used to measure LVEF at rest and their agreement (neither considered as a reference method) was assessed by Bland–Altman plots: LVEF difference (myocardial perfusion scintigraphy minus echocardiography ) against average LVEF ( MPS + Echo 2 ). Results Of patients who had myocardial perfusion scintigraphy and echocardiography performed within six months, mean LVEF difference = +1.1% (95% limits of agreement: −19.3 to +21.6) in men but +10.9% (−10.7 to +32.5) in women. LVEF difference diverged from zero marginally in men (mean difference = +1.1, 95%CI = +0.1 to +2.1, p = 0.028) but more in women (+10.9, +9.8 to +12.1, p

Background Left ventricular ejection fraction (LVEF) is generally measured by echocardiography (Echo) but is increasingly available with myocardial perfusion scintigraphy (MPS). With MPS the threshold of LVEF below which there is a risk for myocardial infarct or sudden cardiac death is higher for women (51%) than for men (43%) [Shahir T, et al. J Nucl Cardiol 2006;13:495–506]. We tested the hypothesis that such a sex difference may also occur with Echo and MPS, by comparing LVEF measured by both methods. Participants and setting: A total of 1141 patients underwent cardiac assessment, including resting LVEF measured both by MPS and Echo. Of these 743 patients (404 men, mean age=67.7±SD=12.3 yr; 339 women, 67.7±11.7 yr) had separate examinations within 6months and their data used for analysis. A subset of 327 of these patients (181 men, 68.8±12.1 yr; 146 women, 66.4±12.1 yr) had examinations at a shorter time apart (within 1month) and were additionally analysed as this sub-group. All examinations were at Ashford & St Peter’s NHS Foundation Trust between 30–11–2012 and 30–05–2017 (figure 1). Methods The rest MPS was performed with the injection of 99mTc-tetrofosmin (600–1000MBq). Images were obtained using dual-head SPECT cameras (Siemens Symbia S, Erlangen, Germany) and LVEF was determined from gated images. The rest Echo was performed with a high-end 2-dimensional echocardiographic unit (Sonos 5500, Andover, Mass., US or Vingmed System V, Horten, Norway) and images were acquired with standard parasternal, short-axis and apical views and LVEF was calculated by the modified Simpson’s biplane disks method. Agreement between MPS and Echo (neither considered as a reference method) was assessed by Bland-Altman plots: LVEF difference (MPS minus Echo) against average LVEF ((MPS+Echo)/2). Data are presented as means and ±SD. Results Of patients who had MPS and Echo examinations within 6months, mean LVEF difference was +1.1% (95% limits of agreement (±2SD)=-19.3 to +21.6) in men (figure 2a) but +10.9% (-10.7 to +32.5) in women (figure 2b). A one-tailed t-test showed LVEF difference diverged from zero only marginally in men (mean difference=+1.1, 95%CI=+0.1 to +2.1, p=0.028) but more in women (+10.9, +9.8 to +12.1, p Conclusion Caution should be taken when interpreting LVEF measured by different techniques due to their wide limits of agreement and systematic bias, more markedly in women. Our data however cannot provide an underlying explanation for these differences but physiological and anatomical differences between men and women may contribute, e.g. cardiac morphology, haemodynamics and body habitus. Conflict of Interest None

AIMS: We studied the relationship between myofilament Ca(2+) sensitivity and troponin I (TnI) phosphorylation by protein kinase A at serines 22/23 in human heart troponin isolated from donor hearts and from myectomy samples from patients with hypertrophic obstructive cardiomyopathy (HOCM). METHODS AND RESULTS: We used a quantitative in vitro motility assay. With donor heart troponin, Ca(2+) sensitivity is two- to three-fold higher when TnI is unphosphorylated. In the myectomy samples from patients with HOCM, the mean level of TnI phosphorylation was low: 0.38 ± 0.19 mol Pi/mol TnI compared with 1.60 ± 0.19 mol Pi/mol TnI in donor hearts, but no difference in myofilament Ca(2+) sensitivity was observed. Thus, troponin regulation of thin filament Ca(2+) sensitivity is abnormal in HOCM hearts. HOCM troponin (0.29 mol Pi/mol TnI) was treated with protein kinase A to increase the level of phosphorylation to 1.56 mol Pi/mol TnI. No difference in EC(50) was found in thin filaments containing high and low TnI phosphorylation levels. This indicates that Ca(2+) sensitivity is uncoupled from TnI phosphorylation in HOCM heart troponin. Coupling could be restored by replacing endogenous troponin T (TnT) with the recombinant TnT T3 isoform. No difference in Ca(2+) sensitivity was observed if TnI was exchanged into HOCM heart troponin or if TnT was exchanged into the highly phosphorylated donor heart troponin. Comparison of donor and HOCM heart troponin by mass spectrometry and with adduct-specific antibodies did not show any differences in TnT isoform expression, phosphorylation or any post-translational modifications. CONCLUSION: An abnormality in TnT is responsible for uncoupling myofibrillar Ca(2+) sensitivity from TnI phosphorylation in the septum of HOCM patients.

Small ubiquitin-related modifier 1 (SUMO1) is a protein family member involved in post-translational modification resulting in cellular processes such as transcriptional regulation, cell signaling, apoptosis and protein stability. It has been shown that SUMO1 indirectly represses oxidative stress

Rationale: Most sarcomere gene mutations that cause hypertrophic cardiomyopathy are missense alleles that encode dominant negative proteins. The potential exceptions are mutations in the MYBPC3 gene (encoding cardiac myosin-binding protein-C [MyBP-C]), which frequently encode truncated proteins. Objective: We sought to determine whether there was evidence of haploinsufficiency in hypertrophic cardiomyopathy caused by MYBPC3 mutations by comparing left ventricular muscle from patients undergoing surgical myectomy with samples from donor hearts. Methods and Results: MyBP-C protein and mRNA levels were quantitated using immunoblotting and RT-PCR. Nine of 37 myectomy samples had mutations in MYBPC3 : 2 missense alleles (Glu258Lys, Arg502Trp) and 7 premature terminations. No specific truncated MyBP-C peptides were detected in whole muscle homogenates of hypertrophic cardiomyopathy tissue. However, the overall level of MyBP-C in myofibrils was significantly reduced ( P MYBPC3 mutation: 0.76±0.03 compared with 1.00±0.05 in donor and 1.01±0.06 in non- MYBPC3 mutant myectomies. Conclusions: The absence of any detectable truncated MyBP-C argues against its incorporation in the myofiber and any dominant negative effect. In contrast, the lowered relative level of full length protein in both truncation and missense MYBPC3 mutations argues strongly that haploinsufficiency is sufficient to cause the disease.

Many of the links between the genotype and phenotype in hypertrophic cardiomyopathy remain unexplained. In this unique longitudinal study we have investigated a patient with classical clinical phenotypic features of hypertrophic obstructive cardiomyopathy, with a known mutation in MYBPC3, the most commonly affected gene in this disease. By collecting cardiac tissue from the patient at the time of surgical myectomy for relief of left ventricular outflow tract obstruction, we have been able to examine the structure of the myocytes and the functional differences that occur in MyBP-C mutated HCM cardiac tissue from single protein level, onto single cardiomyocyte contractility, through to whole organ function as assessed clinically by echocardiography.

Phosphorylation of myosin binding protein C (MyBP-C) was investigated in intraventricular septum samples taken from patients with hypertrophic cardiomyopathy undergoing surgical septal myectomy. These samples were compared with donor heart muscle, as a well-characterised control tissue, and with end-stage failing heart muscle. MyBP-C was partly purified from myofibrils using a modification of the phosphate-EDTA extraction of Hartzell and Glass. MyBP-C was separated by SDS-PAGE and stained for phosphoproteins using Pro-Q Diamond followed by total protein staining using Coomassie Blue. Relative phosphorylation level was determined from the ratio of Pro-Q Diamond to Coomassie Blue staining of MyBP-C bands as measured by densitometry. We compared 9 myectomy samples and 9 failing heart samples with 9 donor samples. MyBP-C phosphorylation in pathological muscle was lower than in donor (myectomy 40 +/- 2% of donor, P

Despite developments in therapeutic and diagnostic technologies, the global burden of atherosclerotic coronary artery disease is increasing. Intravascular imaging has become an invaluable adjunct to percutaneous coronary intervention. Optical coherence tomography (OCT) is a catheter based invasive imaging system that uses light instead of ultrasound to produce high resolution in vivo images of coronary arteries and deployed stents. The technical aspects of intracoronary image acquisition, advantages, disadvantages of use and current applications of OCT in coronary atherosclerosis are discussed.

Heart failure is a common condition and becoming more common. Many treatments have been evaluated in large clinical trials during the last few decades. These trials can be criticized but do provide an evidence base for treatment, probably greater than for most other clinical conditions. Weaknesses exist in relation to age, ejection fraction, and gender. Most trials have included patients 10 years younger than those in the general population. There is little evidence for efficacy in patients with a near normal ejection fraction. Few women have been included in trials.

Myocardial reperfusion injury has been identified as a key determinant of myocardial infarct size in patients undergoing percutaneous or surgical interventions. Although the molecular mechanisms underpinning reperfusion injury have been elucidated, attempts at translating this understanding into clinical benefit for patients undergoing cardiac interventions have produced mixed results. Ischemic conditioning has been applied before, during, or after an ischemic insult to the myocardium and has taken the form of local induction of ischemia or ischemia of distant tissues. Clinical studies have confirmed the safety of differing conditioning techniques, but the benefit of such techniques in reducing hard clinical event rates has produced mixed results. The aim of this article is to review the role of ischemic conditioning in patients undergoing percutaneous and surgical coronary revascularization.

We present a case of a 76-year-old man with ischemic cardiomyopathy. Cardiac magnetic resonance imaging demonstrated severe left ventricular (LV) impairment with possibility of scar formation. Cardiac resynchronization therapy was employed with the aid of a novel quadripolar LV lead. The quadripolar LV lead can be programmed for 10 different pacing configurations, allowing the electrophysiologist freedom to optimize the vector around scar and also avoid phrenic nerve stimulation without the requirement of LV lead repositioning. (PACE 2013; 36:e45–e47)

Background— Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca 2+ sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca 2+ -regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation. Methods and Results— Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca 2+ -activated force was similar in cTnC G159D and donor myocytes, but the Ca 2+ sensitivity of cTnC G159D myocytes was higher (EC 50 G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca 2+ sensitivity (EC 50 G159D/donor=0.55�0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca 2+ sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca 2+ sensitivity was increased (EC 50 P/unP=4.7�1.9) but not with cTnC G159D troponin (EC 50 P/unP=1.2�0.1). Conclusions— We propose that uncoupling of the relationship between phosphorylation and Ca 2+ sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation.

AIM: The aim of the study was to compare the functional and structural properties of the motor protein, myosin, and isolated myocyte contractility in heart muscle excised from hypertrophic cardiomyopathy patients by surgical myectomy with explanted failing heart and non-failing donor heart muscle. METHODS: Myosin was isolated and studied using an in vitro motility assay. The distribution of myosin light chain-1 isoforms was measured by two-dimensional electrophoresis. Myosin light chain-2 phosphorylation was measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using Pro-Q Diamond phosphoprotein stain. RESULTS: The fraction of actin filaments moving when powered by myectomy myosin was 21% less than with donor myosin (P = 0.006), whereas the sliding speed was not different (0.310 +/- 0.034 for myectomy myosin vs. 0.305 +/- 0.019 microm/s for donor myosin in six paired experiments). Failing heart myosin showed 18% reduced motility. One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M). In myectomy myosin, the level of atrial light chain-1 relative to ventricular light chain-1 was 20 +/- 5% compared with 11 +/- 5% in donor heart myosin and the level of myosin light chain-2 phosphorylation was decreased by 30-45%. Isolated cardiomyocytes showed reduced contraction amplitude (1.61 +/- 0.25 vs. 3.58 +/- 0.40%) and reduced relaxation rates compared with donor myocytes (TT(50%) = 0.32 +/- 0.09 vs. 0.17 +/- 0.02 s). CONCLUSION: Contractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation. The presence of a myosin heavy chain mutation causing hypertrophic cardiomyopathy can be predicted from a simple functional assay.

Introduction High sensitivity troponin assays (Hs-Tn) are part of the diagnostic criteria for identifying myocardial infarction, in the presence of ischaemic chest pain, with or without ECG changes. However, the increased sensitivity of the new generation HS-Tn assays has come at the expense of a marked reduction in specificity, which is further confounded when sampled inappropriately in the absence of clinical features suggestive of an acute coronary syndrome (ACS). We audited the utilisation of HS-Tn in both the emergency and acute medical departments at our institution. Methods A retrospective observational audit was performed before and after formal education was delivered to the hospital regarding use of HS-TnI, between October 2013 and July 2014. The audit was assessed against current European Society of Cardiology guidelines in the use of HS-Tn in diagnosing ACS. The main parameters assessed were presence of symptoms or ECG findings in keeping with an ischaemic-episode and the concordance of the initial diagnosis with the subsequent cardiologist-guided diagnosis. Results Ninety-six consecutive patients were sampled in the two audit cycles (50 in the initial cohort and 46 in the re-audit. All patients had HS-TnI assayed on admission and initial treatment with anti-platelet and anti-thrombotic therapy commenced. 46% (n = 23/50) (Figure 1) vs. 67% (n = 31/46) sampled in the presence of chest pain +/- ECG changes. 8% (n = 4/50) vs. 11% (n = 5/46) were treated for an ACS following cardiology review, with 92% (n = 46/50) vs. 89% (n = 41/46), having no evidence of ACS. 52% (n = 26/46) (Figure 2) vs. 34% (14/41) of the cohorts had an elevated HS-TnI (>0.04 mcg/L), attributable to concurrent sepsis, renal dysfunction or tachy-arrhythmias. Conclusion HS-Tn is frequently performed in the absence of clinical features or clinical suspicion of an ACS, reducing the specificity of the test. There are important clinical and financial implications with the inappropriate use of Troponin assays outside of the context of a clinical suspicion of an ACS. Improved education and adherence to guidelines is paramount in improving clinical and diagnostic accuracy of this cardiac biomarker in identifying ACS.

We made quantitative measurements of phosphorylation in troponin isolated from 6 non-failing donor hearts and 6 explanted hearts with end-stage heart failure in SDS-PAGE gels using Pro-Q Diamond phosphoprotein stain. The troponin T phosphorylation level was the same in troponin from failing and non-failing heart (3.1 mol Pi/mol). However, troponin I phosphorylation was significantly lower in failing (0.37+/-0.18 mol Pi/mol) compared with non-failing heart troponin (2.25+/-0.36 mol Pi/mol). Levels of troponin I PKA-dependent phosphorylation, measured with a phosphoserine 23/24-specific antibody, were also significantly lower in failing heart troponin (0.19+/-0.06 mol Pi/mol) compared to non-failing troponin (1.14+/-0.09 mol Pi/mol). We calculate that there is phosphorylation in addition to serine 23/24 of 1.11+/-0.34 mol Pi/mol in non-failing reduced to 0.18+/-0.17 mol Pi/mol in failing heart troponin, attributed to phosphorylation on the PKC sites. To test for the functional role of troponin I phosphorylation, the native troponin I from either non-failing or failing heart troponin was exchanged for a recombinant (unphosphorylated) human cardiac troponin I. Thin filament Ca(2+)-regulatory function was studied with the quantitative in vitro motility assay: thin filaments containing the replaced troponin I resulted in a failing phenotype of a 17-26% reduced sliding speed and an increased Ca(2+)-sensitivity relative to non-failing troponin (EC(50) TnI-exchanged/non-failing=0.57, p0.001). When exchanged with troponin I phosphorylated with PKA motility parameters reverted to a pattern indistinguishable from non-failing troponin (p=0.35-0.75). We suggest that changes in troponin function can account for the contractile abnormality in failing heart muscle and that the functional changes in troponin are due to reduced phosphorylation of troponin I at the PKA sites.

We report on a 2-year-old male child with both nemaline myopathy and hypertrophic cardiomyopathy (HCM). Sequencing of the ACTA1 gene showed a "de novo" missense heterozygous mutation a>g in exon 7 (Lys336Glu). Two-dimensional electrophoresis showed 28% mutant actin present in his muscle biopsy. Actin was isolated from the muscle biopsy and examined by in vitro motility assay. The sliding speed was 13+/-3% less than normal and the affinity of actin for the Z-line protein alpha-actinin was reduced 10 fold. This is the first report on a hypertrophic cardiomyopathy associated with nemaline myopathy and an ACTA1 mutation.

Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca2+-activated force was much lower in HCM myocytes (14 ± 1 kN/m2) than in donor myocytes (23 ± 3 kN/m2; P, Research Highlights ► The passive stiffness of skinned HCM cardiac myocytes was similar to that of normal (donor) myocytes. ► Maximum Ca-activated force production was reduced by 40% in HCM vs donor myocytes. ► This loss of force could contribute to systolic dysfunction in HCM hearts. ► Myofibrillar Ca sensitivity was higher in HCM than in donor myocytes. ► The enhanced Ca sensitivity could compensate for the smaller maximum force but would tend to cause diastolic dysfunction. ► These characteristics were common to all HCM patients studied, suggesting the changes were secondary consequence of the underlying genetic variants.

Sarcomeric mutations in MyBPC that cause hypertrophic cardiomyopathy (HCM) may act as dominant negative alleles by encoding ‘poison polypeptides'or as null alleles resulting in haploinsufficiency. To resolve this we have studied left ventricular muscle samples from patients undergoing surgical myectomy for obstructive HCM and compared these with samples from non-failing (donor) heart muscle. Seven out of 27 myectomy samples were found to contain mutations in MyBP-C: two previously described missense alleles (Glu258Lys, Arg502Trp) and five premature terminations (truncating in domains C3, C5, C7 [x2], C10). Western blots were performed using an antibody shown to recognise specifically the N-terminal region (C0-C2) of MyBPC. MyBPC content was quantified by ELC and densitometry and normalised to staining with an anti-actin antibody.No truncated peptides were detected in whole muscle homogenates, or the myofibrillar fraction, of HCM tissue (including in overloaded gels). However, the overall level of MyBP-C in myofibrils was reduced by 24±4% in myofibrils from tissue containing a MyBP-C mutation: 0.76+/-0.04 (n=39) vs 1.00+/-0.05 in non-failing (n=19)* and 1.01+/-0.05 (n=24) in non-MyBPC mutant myectomies. *p=0.0005. Four of the myectomy samples individually showed statistically significant differences from the non-failing group; these included both truncation and missense samples.The absence of detectable lower molecular weight protein suggests that the truncated MyBPC proteins are degraded, arguing against their incorporation in the myofibre and any dominant negative effect. In contrast, the lowered relative level of full length MyBPC in the myofibre argues strongly for haploinsufficiency as the disease mechanism (potentially for missense as well as truncation alleles). Previous work on partial extraction of MyBPC suggests that lowered MyBPC stoichiometry would be expected to alter muscle function.