Distinct Metabolomic Signatures in Preclinical and Obstructive Hypertrophic Cardiomyopathy.

Hypertrophic Cardiomyopathy (HCM) is a common inherited heart disease with poor risk prediction due to incomplete penetrance and a lack of clear genotype-phenotype correlations. Advanced imaging techniques have shown altered myocardial energetics already in preclinical gene variant carriers. To determine whether disturbed myocardial energetics with the potential to serve as biomarkers are also reflected in the serum metabolome, we analyzed the serum metabolome of asymptomatic carriers in comparison to healthy controls and obstructive HCM patients (HOCM). We performed non-quantitative direct-infusion high-resolution mass spectrometry-based untargeted metabolomics on serum from fasted asymptomatic gene variant carriers, symptomatic HOCM patients and healthy controls (n = 31, 14 and 9, respectively). Biomarker panels that discriminated the groups were identified by performing multivariate modeling with gradient-boosting classifiers. For all three group-wise comparisons we identified a panel of 30 serum metabolites that best discriminated the groups. These metabolite panels performed equally well as advanced cardiac imaging modalities in distinguishing the groups. Seven metabolites were found to be predictive in two different comparisons and may play an important role in defining the disease stage. This study reveals unique metabolic signatures in serum of preclinical carriers and HOCM patients that may potentially be used for HCM risk stratification and precision therapeutics.

Metabolomics in Severe Aortic Stenosis Reveals Intermediates of Nitric Oxide Synthesis as Most Distinctive Markers.

BACKGROUND: Calcific aortic valve disease (CAVD) is a rapidly growing global health problem with an estimated 12.6 million cases globally in 2017 and a 112% increase of deaths since 1990 due to aging and population growth. CAVD may develop into aortic stenosis (AS) by progressive narrowing of the aortic valve. AS is underdiagnosed, and if treatment by aortic valve replacement (AVR) is delayed, this leads to poor recovery of cardiac function, absence of symptomatic improvement and marked increase of mortality. Considering the current limitations to define the stage of AS-induced cardiac remodeling, there is need for a novel method to aid in the diagnosis of AS and timing of intervention, which may be found in metabolomics profiling of patients. METHODS: Serum samples of nine healthy controls and 10 AS patients before and after AVR were analyzed by untargeted mass spectrometry. Multivariate modeling was performed to determine a metabolic profile of 30 serum metabolites which distinguishes AS patients from controls. Human cardiac microvascular endothelial cells (CMECs) were incubated with serum of the AS patients and then stained for ICAM-1 with Western Blot to analyze the effect of AS patient serum on endothelial cell activation. RESULTS: The top 30 metabolic profile strongly distinguishes AS patients from healthy controls and includes 17 metabolites related to nitric oxide metabolism and 12 metabolites related to inflammation, in line with the known pathomechanism for calcific aortic valve disease. Nine metabolites correlate strongly with left ventricular mass, of which three show reversal back to control values after AVR. Western blot analysis of CMECs incubated with AS patient sera shows a significant reduction (14%) in ICAM-1 in AS samples taken after AVR compared to AS patient sera before AVR. CONCLUSION: Our study defined a top 30 metabolic profile with biological and clinical relevance, which may be used as blood biomarker to identify AS patients in need of cardiac surgery. Future studies are warranted in patients with mild-to-moderate AS to determine if these metabolites reflect disease severity and can be used to identify AS patients in need of cardiac surgery.

Myocardial adaptation after surgical therapy differs for aortic valve stenosis and hypertrophic obstructive cardiomyopathy.

Surgical therapies in aortic valve stenosis (AVS) and hypertrophic obstructive cardiomyopathy (HOCM) aim to relief intraventricular pressure overload and improve clinical outcome. It is currently unknown to what extent myocardial adaptation concurs with restoration of intraventricular pressures, and whether this is similar in both patient groups. The aim of this study was to investigate changes in myocardial adaptation after surgical therapies for AVS and HOCM. Ten AVS and ten HOCM patients were enrolled and underwent cardiac magnetic resonance cine imaging and myocardial tagging prior to, and 4 months after aortic valve replacement (AVR) and septal myectomy, respectively. Global left ventricular (LV) analyses were derived from cine images. Circumferential strain was assessed from myocardial tagging images at the septal and lateral wall of the mid ventricle. Pressure gradients significantly decreased in both AVS and HOCM after surgery (p < 0.01), with a concomitant decrease in left atrial volume (p < 0.05) suggesting lower diastolic filling pressures. Also, LV volumes, mass and septal wall thickness decreased in both, but to a larger extent in AVS than in HOCM patients. AVR improved wall thickening (p < 0.05) and did not change systolic strain rate. Myectomy did not affect wall thickening and reduced septal systolic strain rate (p = 0.03). Both AVR and myectomy induced positive structural remodeling in line with a reduction of pressure overload. A concomitant recovery in systolic function however was found in AVR only. The systolic functional deterioration in HOCM patients seems to be inherent to myectomy and the ongoing and irreversible disease.

Cardiac Myosin Binding Protein-C Autoantibodies are Potential Early Indicators of Cardiac Dysfunction and Patient Outcome in Acute Coronary Syndrome.

The degradation and release of cardiac myosin binding protein-C (cMyBP-C) upon cardiac damage may stimulate an inflammatory response and autoantibody (AAb) production. We determined whether the presence of cMyBP-C-AAbs associated with adverse cardiac function in CVD patients. Importantly, cMyBP-C-AAbs were significantly detected in ACS patient sera upon arrival to the emergency department, particularly in STEMI patients. Patients positive for cMyBP-C-AAbs had a reduced LVEF and elevated levels of clinical biomarkers of MI. We conclude that cMyBP-C-AAbs may serve as early predictive indicators of deteriorating cardiac function and patient outcome in ACS patients prior to the infarction.

Disease Stage-Dependent Changes in Cardiac Contractile Performance and Oxygen Utilization Underlie Reduced Myocardial Efficiency in Human Inherited Hypertrophic Cardiomyopathy.

BACKGROUND: Reduced myocardial efficiency represents a target for therapy in hypertrophic cardiomyopathy although therapeutic benefit may depend on disease stage. Here, we determined disease stage-dependent changes in myocardial efficiency and effects of myectomy surgery. METHODS AND RESULTS: Myocardial external efficiency (MEE) was determined in 27 asymptomatic mutation carriers (genotype positive/phenotype negative), 10 patients with hypertrophic obstructive cardiomyopathy (HOCM), 10 patients with aortic valve stenosis, and 14 healthy individuals using [11C]-acetate positron emission tomography and cardiovascular magnetic resonance imaging. Follow-up measurements were performed in HOCM and aortic valve stenosis patients 4 months after surgery. External work did not differ in HOCM compared with controls, whereas myocardial oxygen consumption was lower in HOCM. Because of a higher cardiac mass, total cardiac oxygen consumption was significantly higher in HOCM than in controls and genotype positive/phenotype negative. MEE was significantly lower in genotype positive/phenotype negative than in controls (28±6% versus 42±6%) and was further decreased in HOCM (22±5%). In contrast to patients with aortic valve stenosis, MEE was not improved in patients with HOCM after surgery, which was explained by opposite changes in the septum (decrease) and lateral (increase) wall. CONCLUSIONS: Different mechanisms underlie reduced MEE at the early and advanced stage of hypertrophic cardiomyopathy. The initial increase and subsequent reduction in myocardial oxygen consumption during disease progression indicates that energy deficiency is a primary mutation-related event, whereas mechanisms secondary to disease remodeling underlie low MEE in HOCM. Our data highlight that the benefit of therapies to improve energetic status of the heart may vary depending on the disease stage and that treatment should be initiated before cardiac remodeling.

Left ventricular outflow tract gradient is associated with reduced capillary density in hypertrophic cardiomyopathy irrespective of genotype.

BACKGROUND: Coronary microvascular dysfunction (CMD) is an important feature of hypertrophic cardiomyopathy (HCM), which contributes negatively to symptoms and long-term outcome. Previous in vivo imaging studies in HCM suggest that left ventricular outflow tract (LVOT) gradient and genetic status are important contributors to CMD. CMD may be caused by reduced capillary density. Here, we investigated whether a reduction in capillary density is related to genetic status or LVOT gradient severity in an in vitro study of HCM cardiac samples. METHODS: Using immunofluorescence microscopy, we analysed capillaries (Cap) and cardiomyocytes (CM) in myectomy specimens from 18 HCM patients with maximum left ventricular (LV) wall thickness ≥15 mm. All subjects exhibited significant LVOT obstruction, necessitating septal myectomy. In addition, control myocardium from the LV septal wall was collected at autopsy of 6 individuals that suffered a noncardiac death. RESULTS: CM area was higher in patients with HCM compared to controls. Capillary density was significantly lower in patients with HCM compared with controls (1425 ± 262 vs. 2543 ± 509 Cap/mm(2) , P < 0·001), as was the number of Cap per CM corrected for CM area (2·2 ± 0·5 vs. 4·2 ± 0·9 Cap/CM area, P < 0·001). Capillary density did not differ between genotype-negative and genotype-positive HCM patients at similar resting LVOT gradients. A significant correlation was present between resting LVOT gradient and CM area (r = 0·73, P < 0·001), capillary density (r = -0·74, P < 0·001) and the number of Cap per CM corrected for CM area (r = -0·82, P < 0·001). CONCLUSIONS: Our data indicate that LVOT gradient, rather than genetic status, is associated with reduced capillary density in HCM.

Myocardial efficiency is an important determinant of functional improvement after aortic valve replacement in aortic valve stenosis patients: a combined PET and CMR study.

AIMS: The pathophysiology underlying aortic valve stenosis (AVS)-induced cardiac dysfunction and reduced exercise capacity is unclear. We hypothesize that improvement of myocardial external efficiency (MEE)--the ratio between external work and myocardial oxygen consumption (MVO2)--underlies functional improvement of AVS patients after aortic valve replacement (AVR). Therefore, the aim of this proof-of-concept study was to investigate whether myocardial efficiency is reduced in patients with cardiac hypertrophy caused by AVS and to assess the effect of AVR on myocardial efficiency in relation to exercise capacity. METHODS AND RESULTS: Echocardiography, cardiopulmonary exercise test, [(11)C]-acetate positron emission tomography and cardiovascular magnetic resonance imaging were performed in 10 AVS patients prior to (pre-AVR) and 4 months after AVR (post-AVR). Fourteen healthy individuals served as control group. MEE was significantly lower in pre-AVR patients (32 ± 7%) than in controls (49 ± 6%). AVR significantly decreased left ventricle mass and MVO2. Also, external work significantly decreased post-AVR reaching similar values as in controls. AVR significantly improved MEE from 32 ± 7 to 37 ± 5% (P = 0.02). Moreover, significant correlations were present between the AVR-induced increase in MEE and changes in both exercise work (r = 0.74, P = 0.01) and peak VO2 (r = 0.67, P = 0.03). However, four AVS patients did not show improved MEE, which was associated with no or minimal improvement in exercise parameters. CONCLUSION: MEE is significantly reduced in patients with AVS-induced hypertrophy. Improved MEE is an important predictor of AVR-induced improvement of exercise capacity in AVS patients. Future investigation is needed to confirm our observations in a large prospective, multicenter clinical trial.

Sexual dimorphic response to exercise in hypertrophic cardiomyopathy-associated MYBPC3-targeted knock-in mice.

Hypertrophic cardiomyopathy (HCM), the most common genetic cardiac disorder, is frequently caused by mutations in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Moreover, HCM is the leading cause of sudden cardiac death (SCD) in young athletes. Interestingly, SCD is more likely to occur in male than in female athletes. However, the pathophysiological mechanisms leading to sex-specific differences are poorly understood. Therefore, we studied the effect of sex and exercise on functional properties of the heart and sarcomeres in mice carrying a MYBPC3 point mutation (G > A transition in exon 6) associated with human HCM. Echocardiography followed by isometric force measurements in left ventricular (LV) membrane-permeabilized cardiomyocytes was performed in wild-type (WT) and heterozygous (HET) knock-in mice of both sex (N = 5 per group) in sedentary mice and mice that underwent an 8-week voluntary wheel-running exercise protocol. Isometric force measurements in single cardiomyocytes revealed a lower maximal force generation (F max) of the sarcomeres in male sedentary HET (13.0 ± 1.1 kN/m(2)) compared to corresponding WT (18.4 ± 1.8 kN/m(2)) male mice. Exercise induced a higher F max in HET male mice, while it did not affect HET females. Interestingly, a low cardiac troponin I bisphosphorylation, increased myofilament Ca(2+)-sensitivity, and LV hypertrophy were particularly observed in exercised HET females. In conclusion, in sedentary animals, contractile differences are seen between male and female HET mice. Male and female HET hearts adapted differently to a voluntary exercise protocol, indicating that physiological stimuli elicit a sexually dimorphic cardiac response in heterozygous MYBPC3-targeted knock-in mice.

Sex-dependent pathophysiological mechanisms in hypertrophic cardiomyopathy: implications for rhythm disorders.

Differences in cardiac physiology are seen between men and women in terms of health and disease. Sex differences start to develop at puberty and are maintained during aging. The prevalence of almost all cardiovascular diseases is found to be higher in men than in women, and disease progression tends to be more rapid in male than in female patients. In cohorts of patients with hypertrophic cardiomyopathy (HCM), the most common autosomal inherited cardiac disease, men are overrepresented, suggesting increased penetrance of HCM-causing mutations in male patients. Cardiac remodeling in patients with HCM is higher in men than in women, the same is seen in HCM animal models. Patients with HCM are at increased risk of sudden cardiac death (SCD) and developing rhythm disorders. There seems to be no sex effect on the risk of SCD or arrhythmias in patients with HCM; however, animal studies suggest that certain mutations predispose men to SCD.

Gene-specific increase in the energetic cost of contraction in hypertrophic cardiomyopathy caused by thick filament mutations.

AIMS: Disease mechanisms regarding hypertrophic cardiomyopathy (HCM) are largely unknown and disease onset varies. Sarcomere mutations might induce energy depletion for which until now there is no direct evidence at sarcomere level in human HCM. This study investigated if mutations in genes encoding myosin-binding protein C (MYBPC3) and myosin heavy chain (MYH7) underlie changes in the energetic cost of contraction in the development of human HCM disease. METHODS AND RESULTS: Energetic cost of contraction was studied in vitro by measurements of force development and ATPase activity in cardiac muscle strips from 26 manifest HCM patients (11 MYBPC3mut, 9 MYH7mut, and 6 sarcomere mutation-negative, HCMsmn). In addition, in vivo, the ratio between external work (EW) and myocardial oxygen consumption (MVO2) to obtain myocardial external efficiency (MEE) was determined in 28 pre-hypertrophic mutation carriers (14 MYBPC3mut and 14 MYH7mut) and 14 healthy controls using [(11)C]-acetate positron emission tomography and cardiovascular magnetic resonance imaging. Tension cost (TC), i.e. ATPase activity during force development, was higher in MYBPC3mut and MYH7mut compared with HCMsmn at saturating [Ca(2+)]. TC was also significantly higher in MYH7mut at submaximal, more physiological [Ca(2+)]. EW was significantly lower in both mutation carrier groups, while MVO2 did not differ. MEE was significantly lower in both mutation carrier groups compared with controls, showing the lowest efficiency in MYH7 mutation carriers. CONCLUSION: We provide direct evidence that sarcomere mutations perturb the energetic cost of cardiac contraction. Gene-specific severity of cardiac abnormalities may underlie differences in disease onset and suggests that early initiation of metabolic treatment may be beneficial, in particular, in MYH7 mutation carriers.

Feature tracking compared with tissue tagging measurements of segmental strain by cardiovascular magnetic resonance.

BACKGROUND: Left ventricular segmental wall motion analysis is important for clinical decision making in cardiac diseases. Strain analysis with myocardial tissue tagging is the non-invasive gold standard for quantitative assessment, however, it is time-consuming. Cardiovascular magnetic resonance myocardial feature-tracking (CMR-FT) can rapidly perform strain analysis, because it can be employed with standard CMR cine-imaging. The aim is to validate segmental peak systolic circumferential strain (peak SCS) and time to peak systolic circumferential strain (T2P-SCS) analysed by CMR-FT against tissue tagging, and determine its intra and inter-observer variability. METHODS: Patients in whom both cine CMR and tissue tagging has been performed were selected. CMR-FT analysis was done using endocardial (CMR-FTendo) and mid-wall contours (CMR-FTmid). The Intra Class Correlation Coefficient (ICC) and Pearson correlation were calculated. RESULTS: 10 healthy volunteers, 10 left bundle branch block (LBBB) and 10 hypertrophic cardiomyopathy patients were selected. With CMR-FT all 480 segments were analyzable and with tissue tagging 464 segments.Significant differences in mean peak SCS values of the total study group were present between CMR-FTendo and tissue tagging (-23.8 ± 9.9% vs -13.4 ± 3.3%, p<0.001). Differences were smaller between CMR-FTmid and tissue tagging (-16.4 ± 6.1% vs -13.4 ± 3.3%, p=0.001). The ICC of the mean peak SCS of the total study group between CMR-FTendo and tissue tagging was low (0.19 (95%-CI-0.10-0.49), p=0.02). Comparable results were seen between CMR-FTmid and tissue tagging. In LBBB patients, mean T2P-SCS values measured with CMR-FTendo and CMR-FTmid were 418 ± 66 ms, 454 ± 60 ms, which were longer than with tissue tagging, 376 ± 55 ms, both p<0.05. ICC of the mean T2P-SCS between CMR-FTendo and tissue tagging was 0.64 (95%-CI-0.36-0.81), p<0.001, this was better in the healthy volunteers and LBBB group, whereas the ICC between CMR-FTmid and tissue tagging was lower.The intra and inter-observer agreement of segmental peak SCS with CMR-FTmid was lower compared with tissue tagging; similar results were seen for segmental T2P-SCS. CONCLUSIONS: The intra and inter-observer agreement of segmental peak SCS and T2P-SCS is substantially lower with CMR-FTmid compared with tissue tagging. Therefore, current segmental CMR-FTmid techniques are not yet applicable for clinical and research purposes.

Medical-grade honey kills antibiotic-resistant bacteria in vitro and eradicates skin colonization.

BACKGROUND: Antibiotic resistance among microbes urgently necessitates the development of novel antimicrobial agents. Since ancient times, honey has been used successfully for treatment of infected wounds, because of its antibacterial activity. However, large variations in the in vitro antibacterial activity of various honeys have been reported and hamper its acceptance in modern medicine. METHODS: We assessed the in vitro bactericidal activity of Revamil (Bfactory), a medical-grade honey produced under controlled conditions, and assessed its efficacy for reduction of forearm skin colonization in healthy volunteers in a within-subject-controlled trial. RESULTS: With Bacillus subtilis as a test strain, we demonstrated that the variation in bactericidal activity of 11 batches of medical-grade honey was <2-fold. Antibiotic-susceptible and -resistant isolates of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, and Klebsiella oxytoca were killed within 24 h by 10%-40% (vol/vol) honey. After 2 days of application of honey, the extent of forearm skin colonization in healthy volunteers was reduced 100-fold (P < .001), and the numbers of positive skin cultures were reduced by 76% (P < .001). CONCLUSIONS: Revamil is a promising topical antimicrobial agent for prevention or treatment of infections, including those caused by multidrug-resistant bacteria.