Objectives

This study sought to assess myocardial perfusion and tissue oxygenation during vasodilator stress in patients with overt hypertrophic cardiomyopathy (HCM), as well as in HCM mutation carriers without left ventricular (LV) hypertrophy, and to compare findings to those in athletes with comparable hypertrophy and normal controls.

Background

Myocardial perfusion under vasodilator stress is impaired in patients with HCM. Whether this is associated with impaired myocardial oxygenation and tissue ischemia is unknown. Furthermore, it is not known whether perfusion and oxygenation are impaired in HCM mutation carriers without left ventricular hypertrophy (LVH).

Methods

A total of 27 patients with overt HCM, 10 HCM mutation carriers without LVH, 11 athletes, and 20 healthy controls underwent cardiovascular magnetic resonance (CMR) scanning at 3-T. Myocardial function, perfusion (perfusion reserve index [MPRI]), and oxygenation (blood-oxygen level dependent signal intensity [SI] change) under adenosine stress were assessed.

Results

MPRI was significantly reduced in HCM (1.3 ± 0.1) compared to controls (1.8 ± 0.1, p < 0.001) and athletes (2.0 ± 0.1, p < 0.001), but remained normal in HCM mutation carriers without LVH (1.7 ± 0.1; p = 0.61 vs. controls, p = 0.02 vs. overt HCM). Oxygenation response was attenuated in overt HCM (SI change 6.9 ± 1.4%) compared to controls (18.9 ± 1.4%, p < 0.0001) and athletes (18.7 ± 2.0%, p < 0.001). Interestingly, HCM mutation carriers without LVH also showed an impaired oxygenation response to adenosine (10.4 ± 2.0%; p = 0.001 vs. controls, p = 0.16 vs. overt HCM, p = 0.003 vs. athletes).

Conclusions

In overt HCM, both perfusion and oxygenation are impaired during vasodilator stress. However, in HCM mutation carriers without LVH, only oxygenation is impaired. In athletes, stress perfusion and oxygenation are normal. CMR assessment of myocardial oxygenation has the potential to become a novel risk factor in HCM.

Background

Alterations in cardiac metabolism accompany many diseases of the heart. The advent of cardiac hyperpolarized magnetic resonance spectroscopy (MRS), via dynamic nuclear polarization (DNP), has enabled a greater understanding of the in vivo metabolic changes that occur as a consequence of myocardial infarction, hypertrophy and diabetes. However, all cardiac studies performed to date have focused on rats and larger animals, whereas more information could be gained through the study of transgenic mouse models of heart disease. Translation from the rat to the mouse is challenging, due in part to the reduced heart size (1/10th) and the increased heart rate (50%) in the mouse compared to the rat.

Methods and Results

In this study, we have investigated the in vivo metabolism of [1-13C]pyruvate in the mouse heart. To demonstrate the sensitivity of the method to detect alterations in pyruvate dehydrogenase (PDH) flux, two well characterised methods of PDH modulation were performed; overnight fasting and infusion of sodium dichloroacetate (DCA). Fasting resulted in an 85% reduction in PDH flux, whilst DCA infusion increased PDH flux by 123%. A comparison of three commonly used control mouse strains was performed revealing significant metabolic differences between strains.

Conclusions

We have successfully demonstrated a hyperpolarized DNP protocol to investigate in vivo alterations within the diseased mouse heart. This technique offers a significant advantage over existing in vitro techniques as it reduces animal numbers and decreases biological variability. Thus [1-13C]pyruvate can be used to provide an in vivo cardiac metabolic profile of transgenic mice.

CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD) cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T) in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be insufficient.

AMP-activated protein kinase (AMPK), the key sensor and regulator of cellular energy status, is a heterotrimeric enzyme with multiple isoforms for each subunit (α1/α 2; β1/β2; γ1/γ2/γ3). Mutations in PRKAG2, which encodes the γ2 regulatory subunit, cause a cardiomyopathy characterized by hypertrophy and conduction abnormalities. The two reported PRKAG2 transcript variants, γ2-short and γ2-long (encoding 328 and 569 amino acids respectively), are both widely expressed in adult tissues. We show that both γ2 variants are also expressed during cardiogenesis in mouse embryos; expression of the γ3 isoform was also detected unexpectedly at this stage. As neither γ2 transcript is cardiac specific nor differentially expressed during embryogenesis, it is paradoxical that the disease is largely restricted to the heart. However, a recently annotated γ2 transcript, termed γ2-3B as transcription starts at an alternative exon 3b, has been identified; it is spliced in-frame to exon 4 thus generating a protein of 443 residues in mouse with the first 32 residues being unique. It is increasingly expressed in the developing mouse heart and quantitative PCR analysis established that γ2-3B is the major PRKAG2 transcript (~ 60%) in human heart. Antibody against the novel N-terminal sequence showed that γ2-3B is predominantly expressed in the heart where it is the most abundant γ2 protein. The abundance of γ2-3B and its tissue specificity indicate that γ2-3B may have non-redundant role in the heart and hence mediate the predominantly cardiac phenotype caused by PRKAG2 mutations.

Highlights

► We have identified a novel PRKAG2 transcript of intermediate length (γ2-3B). ► γ2-3B is the most abundant cardiac AMPK γ2 at both mRNA and protein levels. ► Functional changes in AMPK containing γ2-3B may mediate PRKAG2 cardiomyopathy. ► γ2 and γ3 are the early embryonic AMPK γ subuits.

Aims

Increasing energy storage capacity by elevating creatine and phosphocreatine (PCr) levels to increase ATP availability is an attractive concept for protecting against ischaemia and heart failure. However, testing this hypothesis has not been possible since oral creatine supplementation is ineffectual at elevating myocardial creatine levels. We therefore used mice overexpressing creatine transporter in the heart (CrT-OE) to test for the first time whether elevated creatine is beneficial in clinically relevant disease models of heart failure and ischaemia/reperfusion (I/R) injury.

Methods and results

CrT-OE mice were selected for left ventricular (LV) creatine 20–100% above wild-type values and subjected to acute and chronic coronary artery ligation. Increasing myocardial creatine up to 100% was not detrimental even in ageing CrT-OE. In chronic heart failure, creatine elevation was neither beneficial nor detrimental, with no effect on survival, LV remodelling or dysfunction. However, CrT-OE hearts were protected against I/R injury in vivo in a dose-dependent manner (average 27% less myocardial necrosis) and exhibited greatly improved functional recovery following ex vivo I/R (59% of baseline vs. 29%). Mechanisms contributing to ischaemic protection in CrT-OE hearts include elevated PCr and glycogen levels and improved energy reserve. Furthermore, creatine loading in HL-1 cells did not alter antioxidant defences, but delayed mitochondrial permeability transition pore opening in response to oxidative stress, suggesting an additional mechanism to prevent reperfusion injury.

Conclusion

Elevation of myocardial creatine by 20–100% reduced myocardial stunning and I/R injury via pleiotropic mechanisms, suggesting CrT activation as a novel, potentially translatable target for cardiac protection from ischaemia.

Summary

The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.

Highlights

► Cardiac fumarase deletion (cFH1-KO) results in mice with elevated cardiac fumarate ► cFH1-KO is compensated for by amino acid influx into the citric acid cycle ► Nrf2 and its target genes are activated in the hearts of cFH1-KO mice ► Fumarate-related Nrf2 activation is cytoprotective and may be of therapeutic use

Robert Clarke and colleagues conduct a meta-analysis of unpublished datasets to examine the causal relationship between elevation of homocysteine levels in the blood and the risk of coronary heart disease. Their data suggest that an increase in homocysteine levels is not likely to result in an increase in risk of coronary heart disease.

Background

Moderately elevated blood levels of homocysteine are weakly correlated with coronary heart disease (CHD) risk, but causality remains uncertain. When folate levels are low, the TT genotype of the common C677T polymorphism (rs1801133) of the methylene tetrahydrofolate reductase gene (MTHFR) appreciably increases homocysteine levels, so “Mendelian randomization” studies using this variant as an instrumental variable could help test causality.

Methods and Findings

Nineteen unpublished datasets were obtained (total 48,175 CHD cases and 67,961 controls) in which multiple genetic variants had been measured, including MTHFR C677T. These datasets did not include measurements of blood homocysteine, but homocysteine levels would be expected to be about 20% higher with TT than with CC genotype in the populations studied. In meta-analyses of these unpublished datasets, the case-control CHD odds ratio (OR) and 95% CI comparing TT versus CC homozygotes was 1.02 (0.98–1.07; p = 0.28) overall, and 1.01 (0.95–1.07) in unsupplemented low-folate populations. By contrast, in a slightly updated meta-analysis of the 86 published studies (28,617 CHD cases and 41,857 controls), the OR was 1.15 (1.09–1.21), significantly discrepant (p = 0.001) with the OR in the unpublished datasets. Within the meta-analysis of published studies, the OR was 1.12 (1.04–1.21) in the 14 larger studies (those with variance of log OR<0.05; total 13,119 cases) and 1.18 (1.09–1.28) in the 72 smaller ones (total 15,498 cases).

Conclusions

The CI for the overall result from large unpublished datasets shows lifelong moderate homocysteine elevation has little or no effect on CHD. The discrepant overall result from previously published studies reflects publication bias or methodological problems.

Please see later in the article for the Editors' Summary

Editors' Summary

Background

Coronary heart disease (CHD) is the leading cause of death among adults in developed countries. With age, fatty deposits (atherosclerotic plaques) coat the walls of the coronary arteries, the blood vessels that supply the heart with oxygen and nutrients. The resultant restriction of the heart's blood supply causes shortness of breath, angina (chest pains that are usually relieved by rest), and sometimes fatal heart attacks. Many established risk factors for CHD, including smoking, physical inactivity, being overweight, and eating a fat-rich diet, can be modified by lifestyle changes. Another possible modifiable risk factor for CHD is a high blood level of the amino acid homocysteine. Methylene tetrahydofolate reductase, which is encoded by the MTHFR gene, uses folate to break down and remove homocysteine so fortification of cereals with folate can reduce population homocysteine blood levels. Pooled results from prospective observational studies that have looked for an association between homocysteine levels and later development of CHD suggest that the reduction in homocysteine levels that can be achieved by folate supplementation is associated with an 11% lower CHD risk.

Why Was This Study Done?

Prospective observational studies cannot prove that high homocysteine levels cause CHD because of confounding, the potential presence of other unknown shared characteristics that really cause CHD. However, an approach called “Mendelian randomization” can test whether high blood homocysteine causes CHD. A common genetic variant of the MTHFR gene—the C677T polymorphism—reduces MTHFR efficiency so TT homozygotes (individuals in whom both copies of the MTHFR gene have the nucleotide thymine at position 677; the human genome contains two copies of most genes) have 25% higher blood homocysteine levels than CC homozygotes. In meta-analyses (statistical pooling of the results of several studies) of published Mendelian randomized studies, TT homozygotes have a higher CHD risk than CC homozygotes. Because gene variants are inherited randomly, they are not subject to confounding, so this result suggests that high blood homocysteine causes CHD. But what if only Mendelian randomization studies that found an association have been published? Such publication bias would affect this aggregate result. Here, the researchers investigate the association of the MTHFR C677T polymorphism with CHD in unpublished datasets that have analyzed this polymorphism incidentally during other genetic studies.

What Did the Researchers Do and Find?

The researchers obtained 19 unpublished datasets that contained data on the MTHFR C677T polymorphism in thousands of people with and without CHD. Meta-analysis of these datasets indicates that the excess CHD risk in TT homozygotes compared to CC homozygotes was 2% (much lower than predicted from the prospective observational studies), a nonsignificant difference (that is, it could have occurred by chance). When the probable folate status of the study populations (based on when national folic acid fortification legislation came into effect) was taken into account, there was still no evidence that TT homozygotes had an excess CHD risk. By contrast, in an updated meta-analysis of 86 published studies of the association of the polymorphism with CHD, the excess CHD risk in TT homozygotes compared to CC homozygotes was 15%. Finally, in a meta-analysis of randomized trials on the use of vitamin B supplements for homocysteine reduction, folate supplementation had no significant effect on the 5-year incidence of CHD.

What Do These Findings Mean?

These analyses of unpublished datasets are consistent with lifelong moderate elevation of homocysteine levels having no significant effect on CHD risk. In other words, these findings indicate that circulating homocysteine levels within the normal range are not causally related to CHD risk. The meta-analysis of the randomized trials of folate supplementation also supports this conclusion. So why is there a discrepancy between these findings and those of meta-analyses of published Mendelian randomization studies? The discrepancy is too large to be dismissed as a chance finding, suggest the researchers, but could be the result of publication bias—some studies might have been prioritized for publication because of the positive nature of their results whereas the unpublished datasets used in this study would not have been affected by any failure to publish null results. Overall, these findings reveal a serious example of publication bias and argue against the use of folate supplements as a means of reducing CHD risk.

Additional Information

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001177.

The American Heart Association provides information about CHD and tips on keeping the heart healthy; it also provides information on homocysteine, folic acid, and CHD, general information on supplements and heart health, and personal stories about CHD

The UK National Health Service Choices website provides information about CHD, including personal stories about CHD

Information is available from the British Heart Foundation on heart disease and keeping the heart healthy

The US National Heart Lung and Blood Institute also provides information on CHD (in English and Spanish)

MedlinePlus provides links to many other sources of information on CHD (in English and Spanish)

Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)

Objective

The combined thickness of the intima and media of the carotid artery (carotid intima-medial thickness, CIMT) is associated with cardiovascular disease and stroke. Previous studies indicate that carotid intima-medial thickness is a significantly heritable phenotype, but the responsible genes are largely unknown. Hexose-6 phosphate dehydrogenase (H6PDH) is a microsomal enzyme whose activity regulates corticosteroid metabolism in the liver and adipose tissue; variability in measures of corticosteroid metabolism within the normal range have been associated with risk factors for cardiovascular disease. We performed a genetic association study in 854 members of 224 families to assess the relationship between polymorphisms in the gene coding for hexose-6 phosphate dehydrogenase (H6PD) and carotid intima-medial thickness.

Methods

Families were ascertained via a hypertensive proband. CIMT was measured using B-mode ultrasound. Single nucleotide polymorphisms (SNPs) tagging common variation in the H6PD gene were genotyped. Association was assessed following adjustment for significant covariates including “classical” cardiovascular risk factors. Functional studies to determine the effect of particular SNPs on H6PDH were performed.

Results

There was evidence of association between the single nucleotide polymorphism rs17368528 in exon five of the H6PD gene, which encodes an amino-acid change from proline to leucine in the H6PDH protein, and mean carotid intima-medial thickness (p = 0.00065). Genotype was associated with a 5% (or 0.04 mm) higher mean carotid intima-medial thickness measurement per allele, and determined 2% of the population variability in the phenotype.

Conclusions

Our results suggest a novel role for the H6PD gene in atherosclerosis susceptibility.

The metabolic phenotype of the failing heart includes a decrease in phosphocreatine and total creatine concentration [Cr], potentially contributing to contractile dysfunction. Surprisingly, in 32 week old mice over-expressing the myocardial creatine transporter (CrT-OE), we previously demonstrated that elevated [Cr] correlates with left ventricular (LV) hypertrophy and failure. The aim of this study was to determine the temporal relationship between elevated [Cr] and the onset of cardiac dysfunction and to screen for potential molecular mechanisms. CrT-OE mice were compared with wild-type (WT) littermate controls longitudinally using cine-MRI to measure cardiac function and single-voxel 1H-MRS to measure [Cr] in vivo at 6, 16, 32, and 52 weeks of age. CrT-OE mice had elevated [Cr] at 6 weeks (mean 1.9-fold), which remained constant throughout life. Despite this increased [Cr], LV dysfunction was not apparent until 16 weeks and became more pronounced with age. Additionally, LV tissue from 12 to 14 week old CrT-OE mice was compared to WT using 2D difference in-gel electrophoresis (DIGE). These analyses detected a majority of the heart’s metabolic enzymes and identified 7 proteins that were differentially expressed between groups. The most pronounced protein changes were related to energy metabolism: α- and β-enolase were selectively decreased (p<0.05), while the remaining enzymes of glycolysis were unchanged. Consistent with a decrease in enolase content, its activity was significantly lower in CrT-OE hearts (in WT, 0.59±0.02 μmol ATP produced/μg protein/min; CrT-OE, 0.31±0.06; p<0.01). Additionally, anaerobic lactate production was decreased in CrT-OE mice (in WT, 102±3 μmol/g wet myocardium; CrT-OE, 78±13; p=0.02), consistent with decreased glycolytic capacity. Finally, we found that enolase may be regulated by increased expression of the β-enolase repressor transcription factor, which was significantly increased in CrT-OE hearts. This study demonstrates that chronically increased myocardial [Cr] in the CrT-OE model leads to the development of progressive hypertrophy and heart failure, which may be mediated by a compromise in glycolytic capacity at the level of enolase.

Objectives

To further define the role of sarcomere mutations in DCM and associated clinical phenotypes.

Background

Mutations in several contractile proteins contribute to DCM, but definitive evidence for the roles of most sarcomere genes remains limited by the lack of robust genetic support.

Methods

Direct sequencing of 6 sarcomere genes was performed on 334 probands with DCM. A novel D230N missense mutation in the gene encoding α-tropomyosin (TPM1) was identified. Functional assessment was performed using an in vitro reconstituted sarcomere complex to evaluate ATPase regulation and Ca2+ affinity as correlates of contractility.

Results

TPM1 D230N segregated with DCM in two large unrelated families. This mutation altered an evolutionarily conserved residue and was absent in >1000 control chromosomes. In vitro studies demonstrated major inhibitory effects on sarcomere function with reduced Ca2+-sensitivity, maximum activation, and Ca2+ affinity compared to wildtype TPM1. Clinical manifestations ranged from decompensated heart failure or sudden death in those presenting early in life, to asymptomatic left ventricular dysfunction in those diagnosed during adulthood. Notably, several affected infants had remarkable improvement.

Conclusions

Genetic segregation in 2 unrelated families and functional analyses conclusively establish a pathogenic role for TPM1 mutations in DCM. In vitro results demonstrate contrasting effects of DCM and HCM mutations in TPM1, suggesting that specific functional consequences shape cardiac remodeling. Along with prior reports, our data support a distinctive, age-dependent phenotype with sarcomere-associated DCM where presentation early in life is associated with severe, sometimes lethal, disease. These observations have implications for the management of familial DCM.

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 < 0.01), though cross-bridge kinetics (ktr) during maximal Ca2+ activation were 10% faster in HCM myocytes. Myofibrillar Ca2+ sensitivity in HCM myocytes (pCa50 = 6.40 ± 0.05) was higher than for donor myocytes (pCa50 = 6.09 ± 0.02; P < 0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca2+ concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca2+-activated force and high Ca2+ sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients.

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.

Aims

This work investigates the role of myoglobin in mediating the vascular relaxation induced by nitrite. Nitrite, previously considered an inert by-product of nitric oxide metabolism, is now believed to play an important role in several areas of pharmacology and physiology. Myoglobin can act as a nitrite reductase in the heart, where it is plentiful, but it is present at a far lower level in vascular smooth muscle—indeed, its existence in the vessel wall is controversial. Haem proteins have been postulated to be important in nitrite-induced vasodilation, but the specific role of myoglobin is unknown. The current study was designed to confirm the presence of myoglobin in murine aortic tissue and to test the hypothesis that vascular wall myoglobin is important for nitrite-induced vasodilation.

Methods and results

Aortic rings from wild-type and myoglobin knockout mice were challenged with nitrite, before and after exposure to the haem-protein inhibitor carbon monoxide (CO). CO inhibited vasodilation in wild-type rings but not in myoglobin-deficient rings. Restitution of myoglobin using a genetically modified adenovirus both increased vasodilation to nitrite and reinstated the wild-type pattern of response to CO.

Conclusion

Myoglobin is present in the murine vasculature and contributes significantly to nitrite-induced vasodilation.

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.

Author Summary

Heart disease is very common. Some cases of heart disease are strongly influenced by lifestyle and diet, whereas others have a strong genetic component. A certain form of heart failure, known as dilated cardiomyopathy (DCM) quite often runs in families suggesting that a defective gene or genes underlie this disease. We describe a new mouse mutant called “Python” which suffers from a heart disease similar to DCM. We were able to pinpoint the defective gene responsible for the disease. This gene is normally involved in the division of mitochondria, the “power plants” of the cell that generate one of the main energy supplies for the cell. This is a unique model that implicates a new gene and mechanism of disease for further investigation.

Background

It is well established that the immature myocardium preferentially utilises non-oxidative energy-generating pathways. It exhibits low energy-transfer capacity via the creatine kinase (CK) shuttle, reflected in phosphocreatine (PCr), total creatine and CK levels that are much lower than those of adult myocardium. The mechanisms leading to gradually increasing energy transfer capacity during maturation are poorly understood. Creatine is not synthesised in the heart, but taken up exclusively by the action of the creatine transporter protein (CrT). To determine whether this transporter is ontogenically regulated, the present study serially examined CrT gene expression pattern, together with creatine uptake kinetics and resulting myocardial creatine levels, in rats over the first 80 days of age.

Results

Rats were studied during the late prenatal period (-2 days before birth) and 7, 13, 21, 33, 50 and 80 days after birth. Activity of cardiac citrate synthase, creatine kinase and its isoenzymes as well as lactate dehydrogenase (LDH) and its isoenzymes demonstrated the well-described shift from anaerobic towards aerobic metabolism. mRNA levels of CrT in the foetal rat hearts, as determined by real-time PCR, were about 30% of the mRNA levels in the adult rat heart and gradually increased during development. Creatine uptake in isolated perfused rat hearts increased significantly from 3.0 nmol/min/gww at 13 days old to 4.9 nmol/min/gww in 80 day old rats. Accordingly, total creatine content in hearts, measured by HPLC, increased steadily during maturation (30 nmol/mg protein (-2 days) vs 87 nmol/mg protein (80 days)), and correlated closely with CrT gene expression.

Conclusions

The maturation-dependant alterations of CK and LDH isoenzyme activities and of mitochondrial oxidative capacity were paralleled by a progressive increase of CrT expression, creatine uptake kinetics and creatine content in the heart.

Context:

Plasma levels of C-reactive protein (CRP) are independently associated with risk of coronary heart disease, but whether CRP is causally associated with coronary heart disease or merely a marker of underlying atherosclerosis is uncertain.

Objective:

To investigate association of genetic loci with CRP levels and risk of coronary heart disease.

Design, setting and participants:

We first carried out a genome-wide association (n=17,967) and replication study (n=14,747) to identify genetic loci associated with plasma CRP concentrations. Data collection took place between 1989 and 2008 and genotyping between 2003 and 2008. We carried out a Mendelian randomisation study of the most closely associated SNP in the CRP locus and published data on other CRP variants involving a total of 28,112 cases and 100,823 controls, to investigate the association of CRP variants with coronary heart disease. We compared our finding with that predicted from meta-analysis of observational studies of CRP levels and risk of coronary heart disease. For the other loci associated with CRP levels, we selected the most closely associated SNP for testing against coronary heart disease among 14,365 cases and 32,069 controls.

Main outcome measure:

Risk of coronary heart disease.

Results:

Polymorphisms in five genetic loci were strongly associated with CRP levels (% difference per minor allele): SNP rs6700896 in LEPR (−14.7% [95% Confidence Interval {CI}], −17.5 – −11.9, P=1.6×10−21), rs4537545 in IL6R (−10.8% [95% CI, −13.8 – −7.7], P=5.1×10−11), rs7553007 in CRP locus (−20.7% [95% CI, −23.5 – −17.9], P=3.3×10−38), rs1183910 in HNF1A (−13.6% [95% CI, −16.4 – −10.6], P=1.2×10−17) and rs4420638 in APOE-CI-CII (−21.8% [95% CI, −25.4 – −18.1], P=2.1×10−25). Association of SNP rs7553007 in the CRP locus with coronary heart disease gave odds ratio (OR) 0.98 (95% CI, 0.94 – 1.01) per 20% lower CRP. Our Mendelian randomisation study of variants in the CRP locus showed no association with coronary heart disease: OR 1.00 (95% CI, 0.97 – 1.02) per 20% lower CRP, compared with OR 0.94 (95% CI, 0.94 – 0.95) predicted from meta-analysis of the observational studies of CRP levels and coronary heart disease (Z-score −3.45, P<.001). SNPs rs6700896 in LEPR (OR 1.06 [95% CI, 1.02 – 1.09] per minor allele), rs4537545 in IL6R (OR 0.94 [95% CI, 0.91 – 0.97]) and rs4420638 in the APOE-CI-CII cluster (OR 1.16 [95% CI, 1.12 – 1.21]) were all associated with risk of coronary heart disease.

Conclusions:

The lack of concordance between the effect on coronary heart disease risk of CRP genotypes and CRP levels argues against a causal association of CRP with coronary heart disease.

Background

Homocysteine is a sulfur amino acid whose plasma concentration has been associated with the risk of cardiovascular diseases, neural tube defects and loss of cognitive function in epidemiological studies. While genetic variants of MTHFR and CBS are known to influence homocysteine concentration, common genetic determinants of homocysteine remain largely unknown.

Methods and Results

To address this issue comprehensively, we performed a genome wide association analysis, testing 336,469 SNPs in 13,974 healthy Caucasian women. While we confirm association with MTHFR (1p36.22; rs1801133; p=8.1 × 10−35) and CBS (21q22.3; rs6586282; p=3.2 × 10−10), we found novel associations with CPS1 (2q34; rs7422339; p=1.9 × 10−11), MUT (6p12.3; rs4267943; p=2.0 × 10−9), NOX4 (11q14.3; rs11018628; p=9.6 × 10−12) and DPEP1 (16q24.3; rs1126464; p=1.2 × 10−12). The associations at MTHFR, DPEP1 and CBS were replicated in an independent sample from the PROCARDIS study, whereas the association at CPS1 was only replicated among the women.

Conclusions

These associations offer new insights into the biochemical pathways involved in homocysteine metabolism and provide opportunities to better delineate the role of homocysteine in health and disease.

AMP-activated protein kinase (AMPK) is responsible for sensing of the cell’s energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the γ2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. To investigate the polygenetic basis of serum uric acid levels, we conducted a meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent, resulting in identification of 954 SNPs distributed across nine loci that exceeded the threshold of genome-wide significance, five of which are novel. Overall, the common variants associated with serum uric acid levels fall in the following nine regions: SLC2A9 (p = 5.2×10−201), ABCG2 (p = 3.1×10−26), SLC17A1 (p = 3.0×10−14), SLC22A11 (p = 6.7×10−14), SLC22A12 (p = 2.0×10−9), SLC16A9 (p = 1.1×10−8), GCKR (p = 1.4×10−9), LRRC16A (p = 8.5×10−9), and near PDZK1 (p = 2.7×10−9). Identified variants were analyzed for gender differences. We found that the minor allele for rs734553 in SLC2A9 has greater influence in lowering uric acid levels in women and the minor allele of rs2231142 in ABCG2 elevates uric acid levels more strongly in men compared to women. To further characterize the identified variants, we analyzed their association with a panel of metabolites. rs12356193 within SLC16A9 was associated with DL-carnitine (p = 4.0×10−26) and propionyl-L-carnitine (p = 5.0×10−8) concentrations, which in turn were associated with serum UA levels (p = 1.4×10−57 and p = 8.1×10−54, respectively), forming a triangle between SNP, metabolites, and UA levels. Taken together, these associations highlight additional pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia. In addition, these findings strongly support the hypothesis that transport proteins are key in regulating serum uric acid levels.

Author Summary

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. The regulation of serum uric acid levels is under a strong genetic control. This study describes the first meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent. We show that common DNA variants at nine different loci are associated with uric acid concentrations, five of which are novel. These variants are located within the genes coding for organic anion transporter 4 (SLC22A11), monocarboxylic acid transporter 9 (SLC16A9), glucokinase regulatory protein (GCKR), Carmil (LRRC16A), and near PDZ domain-containing 1 (PDZK1). Gender-specific effects are shown for variants within the recently identified genes coding for glucose transporter 9 (SLC2A9) and the ATP-binding cassette transporter (ABCG2). Based on screening of 163 metabolites, we show an association of one of the identified variants within SLC16A9 with DL-carnitine and propionyl-L-carnitine. Moreover, DL-carnitine and propionyl-L-carnitine were strongly correlated with serum UA levels, forming a triangle between SNP, metabolites and UA levels. Taken together, these associations highlight pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia.

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 µm/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 (TT50% = 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.