Β-myosin Heavy Chain Gene Research Articles

MicroRNA-499 improved septic myocardial dysfunction by regulating myosin heavy chain gene axis

To investigate the role and mechanism of microRNA-499 (miR-499) regulating α-myosin heavy chain (α-MHC) and β-myosin heavy chain (β-MHC) gene axis in septic myocardial dysfunction (SMD) and its significance. Sixty healthy adult male Sprague-Dawley (SD) rats were divided into phosphate buffered saline (PBS) control group (PBS group), lipopolysaccharide (LPS) induced SMD model group (LPS group), miR-499 agonist pretreatment group (agomir+LPS group), and miR-499 inhibitor pretreatment group (antagomir+LPS group) by random number table, with 15 rats in each group. SMD rat model was reproduced by intraperitoneal injection of LPS 10 mg/kg. The PBS group was intraperitoneally injected with the same amount of PBS. The two pretreatment groups were injected with agomir 30 mg/kg or antagomir 80 mg/kg through the caudal vein for 3 days, once a day. PBS group and LPS group were not pretreated. Echocardiography was detected 5 hours after LPS injection, and relevant indexes were recorded. The expression of miR-499 in plasma and myocardial tissue was detected by real-time quantitative polymerase chain reaction (qPCR). Western blotting was used to detect the protein expressions of α-MHC and β-MHC in myocardial tissue. Plasma N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of heart failure, was measured by electrochemiluminescence. Compared with the PBS group, the rats in LPS group were depressed. Additionally, LPS down-regulated the level of miR-499 in plasma and myocardial tissue, decreased α-MHC expression in myocardial tissue and up-regulated the expression of β-MHC. Echocardiography showed that left ventricular ejection fraction (LVEF), left ventricle fractional shortening (LVFS), cardiac output (CO), stroke volume (SV) and heart rate (HR) decreased by 49.1%, 59.2%, 48.8%, 39.4% and 15.9%, respectively, and the level of plasma NT-proBNP increased significantly in LPS group, indicating that LPS could induce cardiac dysfunction in rats. Compared with the LPS group, after pretreatment with agomir to overexpress the miR-499, LVEF and LVFS were significantly increased [LVEF: 0.662±0.020 vs. 0.323±0.024, LVFS: (36.16±1.43)% vs. (20.20±1.32)%, both P < 0.01], which suggested that the cardiac function of rats was improved in agomir+LPS group. At the same time, pretreatment with agomir significantly down-regulated the β-MHC protein expression (β-MHC/GAPDH: 0.74±0.04 vs. 2.97±0.34, P < 0.01), significantly up-regulated α-MHC protein expression (α-MHC/GAPDH: 1.59±0.05 vs. 0.74±0.14, P < 0.01), and significantly decreased the plasma NT-proBNP level (ng/L: 114.49±6.85 vs. 334.13±4.36, P < 0.01). After pretreatment with antagomir to inhibit the expression of miR-499, echocardiography showed that LVEF and LVFS were significantly lower than those in the LPS group [LVEF: 0.297±0.021 vs. 0.323±0.024, LVFS: (19.38±1.52)% vs. (21.20±1.32)%, both P < 0.01], which suggested that the cardiac function of rats was significantly inhibited. At the same time, pretreatment with antagomir significantly down-regulated α-MHC protein expression in myocardial tissue (α-MHC/GAPDH: 0.63±0.03 vs. 0.74±0.14, P < 0.01), significantly up-regulated β-MHC protein expression (β-MHC/GAPDH: 3.03±0.47 vs. 2.97±0.34, P < 0.01), and significantly increased the level of plasma NT-proBNP (ng/L: 373.91±4.23 vs. 334.13±4.36, P < 0.05). miR-499 could regulate the expression of α-MHC and β-MHC which improved cardiac dysfunction caused by sepsis. Targeted regulation of miR-499 expression may be an effective way to treat SMD.

Cardiomyopathy Associated with Worsening Intravascular Hemolysis in Transgenic Knock-in Mouse Model of Sickle Cell Disease

Cardiopulmonary complications are the leading cause of death in sickle cell disease (SCD). Cardiac complications, including left ventricular (LV) dysfunction, are prevalent in both young and adult SCD patients. Our studies in the Townes transgenic murine model indicate mice with homozygous SCD (SS) recapitulate the age-related clinical deterioration seen in humans. This is evidenced by the sudden decline in survival of SS mice during the transition from adolescence to adulthood (between the ages of three to six months); we have previously determined that the severity of intravascular hemolysis increases during this transition period in SS mice (Ghosh et al JCI Insight 2016; 1(4): e81090). Hitherto, the cardiac phenotype of this murine model of SCD, which appears the most versatile, has not been defined. In this study, we imaged the hearts of one, three, and six month old Townes SS mice and littermates with sickle cell trait (AS mice) using high resolution ultrasound (Vevo 2100, VisualSonics). Standard indices of LV systolic function, size, and mass, were derived from measurements made on short axis B-mode images of the heart. Ejection fraction and fractional shortening were similar for AS and SS mice, signifying preservation of systolic function in both groups up to 6 months of age. The LV mass, LV internal dimensions (LVID), systolic and diastolic volumes as well as stroke volume significantly increased with age in the SS but not in the AS. Major differences in the cardiac phenotype in SS compared to AS littermates were observed only at six-months. At six months, the SS mice had larger hearts ( 236.5 ± 20.1 mg, n=7, p=0.005) compared to AS littermates (180.2 ± 9.6 mg, n=6). The LVID in both systole (3.1 ± 0.2 mm versus2.1± 0.3, p=0.03) and diastole (4.7 ± 0.2 mmversus 3.9 ± 0.1 mm, p=0.002) were larger in the SS mice, accompanied by higher LV Mass Index (4.6 ± 0.2, n=7 versus 3.1 ± 0.3, n= 6 , p=0.03) indicating LV hypertrophy in the SS mice. RT-PCR analysis of hypertrophy markers in the LV revealed increased expression of Atrial Natriuretic Peptide (ANP, p=0.001) and β-Myosin Heavy Chain (p=0.03) genes in the SS mice compared to AS mice. In agreement with findings in humans, at 6 months, systolic volume (38.4± 6.6 µ L, n=7,versus 17.2±5.3 µ L, n=6, p=0.003) , diastolic volume (101.3± 8.4 µ L, n=7, versus 62.3 ± 4.6 µ L , n=6, p=0.0002), stroke Index (2.2± 0.2, n=7, versus 1.5±0.2, n=6, p=0.03)and cardiac index (1.0±0.1, n=7, versus 0.7±0.1, n=6, p=0.047)were all higher in the six-month SS mice compared to AS littermates. To evaluate vascular inflammation in the heart, P-selectin was measured by ELISA; Compared to AS littermates, SS mice hearts showed a 2-fold higher expression of P-selectin (p=0.009). The severity of anemia and intravascular hemolysis increased with aging in the SS mice. Our study shows progression of LV enlargement and dilation in the Townes SS mice concomitant with increased severity of intravascular hemolysis with aging. Future studies are aimed at delineating the specific molecules and cognate pathways downstream of intravascular hemolysis that promote cardiomyopathy in SCD. DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties.

Mutations in myosin S2 alter cardiac myosin-binding protein-C interaction in hypertrophic cardiomyopathy in a phosphorylation-dependent manner

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disorder primarily caused by mutations in the β-myosin heavy-chain gene. The proximal subfragment 2 region (S2), 126 amino acids of myosin, binds with the C0-C2 region of cardiac myosin-binding protein-C to regulate cardiac muscle contractility in a manner dependent on PKA-mediated phosphorylation. However, it is unknown if HCM-associated mutations within S2 dysregulate actomyosin dynamics by disrupting its interaction with C0-C2, ultimately leading to HCM. Herein, we study three S2 mutations known to cause HCM: R870H, E924K, and E930Δ. First, experiments using recombinant proteins, solid-phase binding, and isothermal titrating calorimetry assays independently revealed that mutant S2 proteins displayed significantly reduced binding with C0-C2. In addition, CD revealed greater instability of the coiled-coil structure in mutant S2 proteins compared with S2Wt proteins. Second, mutant S2 exhibited 5-fold greater affinity for PKA-treated C0-C2 proteins. Third, skinned papillary muscle fibers treated with mutant S2 proteins showed no change in the rate of force redevelopment as a measure of actin–myosin cross-bridge kinetics, whereas S2Wt showed increased the rate of force redevelopment. In summary, S2 and C0-C2 interaction mediated by phosphorylation is altered by mutations in S2, which augment the speed and force of contraction observed in HCM. Modulating this interaction could be a potential strategy to treat HCM in the future.

The role of three-dimensional speckle tracking imaging derived parameters on predicting outcome of hypertrophic cardiomyopathy patients with MYH7 mutations

Objective: To evaluate the cardiac functional changes in hypertrophic cardiomyopathy(HCM) patients with β-myosin heavy chain gene (MYH7) mutations by three-dimensional (3D) speckle tracking imaging(3D-STI) and conventional echocardiography modalities, and then to explore the potential predictors of adverse cardiovascular events in these patients. Methods: A consecutive series of 192 HCM patients admitted in our center from October 2014 to October 2016 were genetically screened to identify MYH7 mutations in this retrospective study. A total of 43 HCM patients with MYH7 mutations were enrolled. The patients were divided into events group(n=13) and no event group(n=30) according to the presence or absence of adverse cardiovascular events(primary and secondary endpoints). All patients were followed up to January 2019 after comprehensive evaluation of 3D-STI, two-dimensional and Doppler echocardiography. The adverse cardiovascular events were recorded. Results: The median follow up time was 1 012 (812, 1 330) days. During follow-up, 13 patients (30.2%) reached endpoints: 6 cases of the primary endpoints(2 cases of sudden cardiac death(SCD), 3 cases of survival after defibrillation, and 1 case of appropriate implantable cardioverter-defibrillator(ICD) discharge); 7 cases of the second endpoints(5 cases of heart failure hospitalization, 1 case of syncope and cardioversion due to supraventricular tachycardia, and 1 case of end-stage HCM). Patients with adverse cardiovascular events had higher prevalence of syncope and risk of SCD, enlarged left atrial volume index(LAVI) and reduced 3D left ventricular global longitudinal train (3D-GLS), as compared to those without adverse events(all P<0.05). The multivariate Cox regression analysis showed that reduced 3D-GLS(HR=0.814, 95%CI 0.663-0.999, P=0.049) was an independent predictor for adverse cardiovascular events. The cutoff value of 3D-GLS≤13.67% was linked with significantly increased risk of adverse cardiovascular events in this patient cohort(AUC=0.753, 95%CI 0.558-0.948, sensitivity 86%, specificity 69%, P<0.05). The Kaplan-Meier analysis indicated that the patients with the 3D-GLS≤ 13.67% faced higher risk of death than those with 3D-GLS>13.67%. Conclusion: 3D-GLS is useful on predicting adverse cardiovascular events in HCM patients with MYH7 mutations.

Novel Variations in β-Myosin Heavy-Chain Gene (β-MYH7) and Its Association in South Indian Women with Cardiomyopathies

Abstract Background Mutations in β-MYH7 gene is a main genetic cause of cardiomyopathy and sudden cardiac arrest, yet the molecular mechanisms have not been fully understood. Objectives To identify variations in β-MYH7 gene and their possible mechanistic role in cardiomyopathies among Indian women. Methods We sequenced all exons and their flanking regions of the β-MYH7 gene in 188 Indian women consisting of 33 hypertrophic cardiomyopathy (HCM), 48 dilated cardiomyopathy (DCM), and 107 healthy controls. Results Our study showed 21 variations in β-MYH7 gene, including 7 novel mutations. In addition, we compared this dataset with our previously studied datasets of seven other sarcomere genes (ACTC, TNNT2, MYL2, MYBPC3, TPM1, TNNI3, and MYL3) and found no causative mutation, confirming the nonexistence of compound heterozygosity. Interestingly, we detected a Val431Met mutation exclusively in patients, and its pathogenicity has been predicted using the protein homology model. In native, Val431 is evolutionarily conserved across many species. In the homology model, mutant Met431 gets further buried in the hydrophobic core by creating an aberrant hydrophobic interaction with Leu352. As a result, it probably reduces the spatial distances between other hydrophobic interactions in the hydrophobic core that may produce steric hindrance and strain. It may lead to deviation in the structure (root means square deviation [RMSD] of ~3.9), and might possibly causing the cardiac remodeling and cardiomyopathy. Conclusion We identified a novel Val431Met mutation, exclusively in patients, and its homology model p.Met431 has profoundly increased the mechanistic understanding of disease specifically in personalized medicine, to block/inverse/diminish the disease phenotype.

CRISPR/Cas9‐mediated Genome Editing in Patient‐Derived iPSC‐Cardiomyocytes Recapitulates an MYH6‐R443P Phenotype in a HLHS Family

Hypoplastic Left Heart Syndrome (HLHS) is a clinically and anatomically severe form of Congenital Heart Disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance, its etiology remains largely unknown. We previously demonstrated that rare variants in the α‐myosin heavy chain (MYH6) gene are significantly enriched in HLHS (p&lt;1×10−5, observed in 10.5% or 20/190 HLHS subjects). Analysis of clinical outcomes showed that survival without cardiac transplant was reduced in HLHS subjects with MYH6 variants (p=6×10−3) as compared to those without MYH6 variants. Transcriptome and protein expression analyses from cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the β‐myosin heavy chain (MYH7) gene in subjects with MYH6 variants. Visualizing α actinin in atria showed intrinsic disrupted sarcomere structure in HLHS patients with MYH6 variants.We observed that patient‐specific induced pluripotent stem cells (iPSCs) were defective in cardiomyogenic differentiation in vitro. Additionally, sarcomere structure in cardiomyocytes derived from iPSCs produced from a family trio (unaffected mother‐affected father‐patient) carrying a variant in MYH6 (MYH6‐R443P) was dysmorphic.We introduced the MYH6‐R443P variant into iPSCs derived from the unaffected parent using CRISPR/cas9 technology and observed that the MYH6‐R443P phenotype in HLHS‐iPSCs was recapitulated; specifically, gene‐edited MYH6‐R443P+/− and MYH6‐R443P−/− iPSCs displayed defective cardiomyogenic differentiation, and sarcomere structure in these cardiomyocytes was dysmorphic. We conclude that the etiology of MYH6‐associated HLHS may be informed utilizing iPSCs.Support or Funding InformationThe Advancing a Healthier Wisconsin Endowment (AHW)Wolfe Family FoundationGreat Milwaukee FoundationChildren's Research InstituteLittle Heart for LifeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypertrophic cardiomyopathy clinical phenotype is independent of gene mutation and mutation dosage

Over 1,500 gene mutations are known to cause hypertrophic cardiomyopathy (HCM). Previous studies suggest that cardiac β-myosin heavy chain (MYH7) gene mutations are commonly associated with a more severe phenotype, compared to cardiac myosin binding protein-C (MYBPC3) gene mutations with milder phenotype, incomplete penetrance and later age of onset. Compound mutations can worsen the phenotype. This study aimed to validate these comparative differences in a large cohort of individuals and families with HCM. We performed genome-phenome correlation among 80 symptomatic HCM patients, 35 asymptomatic carriers and 35 non-carriers, using an 18-gene clinical diagnostic HCM panel. A total of 125 mutations were identified in 14 genes. MYBPC3 and MYH7 mutations contributed to 50.0% and 24.4% of the HCM patients, respectively, suggesting that MYBPC3 mutations were the most frequent cause of HCM in our cohort. Double mutations were found in only nine HCM patients (7.8%) who were phenotypically indistinguishable from single-mutation carriers. Comparisons of clinical parameters of MYBPC3 and MYH7 mutants were not statistically significant, but asymptomatic carriers had high left ventricular ejection fraction and diastolic dysfunction when compared to non-carriers. The presence of double mutations increases the risk for symptomatic HCM with no change in severity, as determined in this study subset. The pathologic effects of MYBPC3 and MYH7 were found to be independent of gene mutation location. Furthermore, HCM pathology is independent of protein domain disruption in both MYBPC3 and MYH7. These data provide evidence that MYBPC3 mutations constitute the preeminent cause of HCM and that they are phenotypically indistinguishable from HCM caused by MYH7 mutations.

Identification of a novel hypertrophic cardiomyopathy-associated mutation using targeted next-generation sequencing.

Hypertrophic cardiomyopathy (HCM), one of the most common forms of myocardial diseases, is the major cause of sudden cardiac death in young adults and competitive athletes. Analyses of gene mutations associated with HCM are valuable for its molecular diagnosis, genetic counseling, and management of familial HCM. To dissect the relationship between the clinical presentation and gene mutations of HCM, the genetic characterizations of 19 HCM-related genes in 18 patients (8 cases from 6 pedigrees with familial HCM and 10 cases without familial HCM) were detected using next-generation sequencing (NGS). As a result, 12 disease-related mutations were identified in the 18 subjects, including 6 single mutations and 3 double mutations [MYBPC3 (p.Gln998Glu) plus TNNI3 (p.Arg145Gly), PRKAG2 (p.Gly100Ser) plus MYBPC3 (p.Lys1209Serfs*28) and TNNI3 (p.Glu124Gln) plus GLA (p.Trp47*)]. The 3 heterozygous double mutations were discovered for the first time in the malignant familial HCM patients. Of the 6 single mutations, a novel mutation was found in tafazzin (TAZ, p.Ile208Val), and a mutation in β-myosin heavy chain gene (MYH7, p.Arg54Gln), which was reported as rare in the general population, was firstly found in one HCM patient. Identification of novel and rare mutations in HCM patients have added new data to the spectrum of gene mutations associated with this disease. These findings provide an essential basis for the molecular diagnosis and better management of family members at risk of familial HCM.

Visualization of Active Transcription Sites in Human Cardiomyocytes Supports the Concept of Burst-Like Transcription of MYH7

Hypertrophic Cardiomyopathy (HCM) has a prevalence of 1 in 500 individuals. More than 1000 mutations in >20 different sarcomeric and some non-sarcomeric proteins have been identified in HCM-patients. In 30-40% the HCM-mutation is found in the β-myosin heavy chain (β-MyHC) gene (MYH7). Until now, a common mechanism triggering HCM is unknown.From previous work of our group on MYH7-mutations we hypothesized that the characteristic HCM-phenotype with hypertrophy, myocyte disarray, and interstitial fibrosis could be a consequence of cell-to-cell functional imbalance due to cell-to-cell variation in the expression of mutated vs. wildtype β-MyHC. Such cell-to-cell variability could result from stochastic, independent burst-like transcription of mutant and wildtype MYH7-alleles.To investigate whether the MYH7-alleles are indeed transcribed in a discontinuous, burst-like manner, we visualized active transcription sites of MYH7 in cardiomyocytes of a patient with β-MyHC-mutation R723G and of a non-transplanted donor heart using fluorescence in situ hybridization. To visualize pre-mRNA in active transcription sites we used two probes sets, each containing 48 single-labeled 20-bases-long oligonucleotides to probe intronic and exonic sequences of MYH7-pre-mRNA, while cytoplasmic MYH7-mRNA was labelled by the exonic probe set. Both probe sets were hybridized to their target sequences in 16 µm thick sections of cardiac samples.Active transcription sites were identified as bright spots inside the nuclei of cardiomyocytes showing co-localization of both probe sets. 26% of the nuclei had no active transcription sites while cytoplasmic MYH7-mRNA was revealed by the exonic probes. Absence of active transcription sites is inconsistent with continuous transcription of the two MYH7-alleles but expected for random burst-like transcription.Model calculations showed that independent, stochastic, burst-like transcription of mutant and wildtype MYH7-alleles can account for the experimentally observed cell-to-cell variability in mutant MYH7-mRNA and functional imbalances.

Impact of MYH6 variants in hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance, its etiology remains largely unknown. The goal of this study was to characterize a risk gene in HLHS and its effect on HLHS etiology and outcome. We performed next-generation sequencing on a multigenerational family with a high prevalence of CHD/HLHS, identifying a rare variant in the α-myosin heavy chain (MYH6) gene. A case-control study of 190 unrelated HLHS subjects was then performed and compared with the 1000 Genomes Project. Damaging MYH6 variants, including novel, missense, in-frame deletion, premature stop, de novo, and compound heterozygous variants, were significantly enriched in HLHS cases (P < 1 × 10−5). Clinical outcomes analysis showed reduced transplant-free survival in HLHS subjects with damaging MYH6 variants (P < 1 × 10−2). Transcriptome and protein expression analyses with cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the β-myosin heavy chain (MYH7) gene in subjects with MYH6 variants (P < 1 × 10−3). We subsequently used patient-specific induced pluripotent stem cells (iPSCs) to model HLHS in vitro. Early stages of in vitro cardiomyogenesis in iPSCs derived from two unrelated HLHS families mimicked the increased expression of MYH7 observed in vivo (P < 1 × 10−2), while revealing defective cardiomyogenic differentiation. Rare, damaging variants in MYH6 are enriched in HLHS, affect molecular expression of contractility genes, and are predictive of poor outcome. These findings indicate that the etiology of MYH6-associated HLHS can be informed using iPSCs and suggest utility in future clinical applications.

MYBPH acts as modifier of cardiac hypertrophy in hypertrophic cardiomyopathy (HCM) patients.

Left ventricular hypertrophy is a risk factor for cardiovascular morbidity and mortality. Hypertrophic cardiomyopathy (HCM) is considered a model disease to study causal molecular factors underlying isolated cardiac hypertrophy. However, HCM manifests with various clinical symptoms, even in families bearing the same genetic defects, suggesting that additional factors contribute to hypertrophy. The gene encoding the cardiac myosin binding protein C (cMYBPC) is one of the most frequently implicated genes in HCM. Recently another myosin binding protein, myosin binding protein H (MYBPH) was shown to function in concert with cMYBPC in regulating cardiomyocyte contraction. Given the similarity in sequence, structure and the critical role MYBPH plays in sarcomere contraction, we proposed that MYBPH may be involved in HCM pathogenesis. Family-based genetic association analysis was employed to investigate the contribution of MYBPH in modifying hypertrophy. Seven single nucleotide polymorphisms and haplotypes in MYBPH were investigated for hypertrophy modifying effects in 388 individuals (27 families), in which three unique South African HCM-causing founder mutations (p.R403W and pA797T in β-myosin heavy chain gene (MYH7) and p.R92W in the cardiac troponin T gene (TNNT2)) segregate. We observed a significant association between rs2250509 and hypertrophy traits in the p.A797T MYH7 mutation group. Additionally, haplotype GGTACTT significantly affected hypertrophy within the same mutation group. MYBPH was for the first time assessed as a candidate hypertrophy modifying gene. We identified a novel association between MYBPH and hypertrophy traits in HCM patients carrying the p.A797T MYH7 mutation, suggesting that variation in MYBPH can modulate the severity of hypertrophy in HCM.

Improvement in fractional shortening in aortic regurgitation rats: cardiac muscle network

Aortic regurgitation (AR), a volume overload to the heart, impairs systolic function. Paroxetine (parox) treatment, a selective serotonin reuptake inhibitor, improves systolic function in AR rats, probably due decreases in the expression of β-myosin heavy chain (βMyHC) gene. An intricate network regulate genes co-expressing microRNAs (miR-208a,-208b and -499) and transcriptional repressors which in turn controls MyHCisoforms content. Thus, we verify the gene expression of those microRNAs and the transcriptional repressor (Thrap1) in AR rats treated with parox. Male Wistar rats (280-300kg) were submitted to sham or AR surgery. Morphofunctional variables of the hearts were analyzed by echocardiograms. Parox (10mg/kg) was administered subcutaneously for 4 weeks. There were 4 groups: AR+parox, AR+saline, Sham+parox and Sham+saline. At week 8 the animals were euthanized for tissue collection and analysis of gene expression by RTq-PCR. Two way repeated measures ANOVA were used for comparisons. Parox treatment preserved the fractional shortening in AR rats. The expression of miR-208a and Thrap 1 were not changed. Though there was a decrease in miR-208b and miR-499 gene expression, which may regulate the decrease of β-MyHC isoform in AR rats treated with parox, explaining the improvement in systolic function. Financial support: FAPESP 2013/11372-6, FAPESP 2013/00742-7

Abstract 11750: Inactivation of Cardiac Foxo1 by Insulin Signaling is Required for Cardiac Function and Suppression of β-Myosin Heavy Chain Gene Expression

Heart failure is a leading cause of morbidity and mortality in the USA and is closely associated with diabetes mellitus. The molecular link between diabetes and heart failure is incompletely understood. We recently demonstrated that insulin receptor substrate 1, 2 (IRS1, 2) are key components of insulin signaling and their dysfunction mediates insulin resistance, resulting in metabolic dysregulation and heart failure. Loss of IRS1 and IRS2 is associated with downstream Akt inactivation and in turn activation of the forkhead transcription factor Foxo1. To determine the role of Foxo1 in control of heart failure in insulin resistance and diabetes, we generated mice lacking Foxo1 gene specifically in the heart. Mice lacking both IRS1 and IRS2 in adult hearts exhibited severe heart failure and a remarkable increase in the β-isoform of myosin heavy chain (β-MHC) gene expression, while deletion of cardiac Foxo1 gene largely prevented the heart failure and resulted in a decrease in β-MHC expression. The effect of Foxo1 deficiency on rescuing cardiac dysfunction was also observed in db/db mice and high-fat diet (HFD) mice. Using cultures of primary ventricular cardiomyocytes, we found that Foxo1 interacts with the promoter region of β-MHC and stimulates gene expression, mediating an effect of insulin that suppresses β-MHC expression. Taken together, our study suggests that Foxo1 has important roles in promoting diabetic cardiomyopathy and controls β-MHC expression in development of cardiac dysfunction. Targeting Foxo1 and its regulation will provide novel strategies in preventing metabolic and myocardial dysfunction and influencing MHC plasticity in diabetes mellitus.

A novel MYH7 Leu1453pro mutation resulting in Laing distal myopathy in an Irish family

Authors describe clinical, pathological, imaging and genetic findings in the first Irish family with Laing distal myopathy in whom a novel mutation in the human slow β-myosin heavy chain (MYH7) gene has been identified. A kindred of 14 over 6 generations included 6 individuals with childhood onset distal lower limb weakness in a scapula-peroneal distribution with subsequent proximal upper and lower limb weakness. Finger extensor weakness especially in the 3rd–5th fingers was present in each and two patients had “hanging big toe” sign. Three patients were non-ambulatory by middle-age. One patient developed cardiomyopathy and two patients had respiratory muscle impairment. Intriguingly, brain white matter lesions and epilepsy were present in three patients. Muscle biopsy revealed fibre-size variation, rimmed vacuoles, mild-extensive central nucleation, redundant and folded sarcolemmal membrane and Z band streaming. Genetic analysis revealed a novel heterozygous mutation in the MYH7 gene in one patient which co-segregated perfectly in the remaining 5 affected members and was absent in six unaffected members.

Cytosolic CARP promotes angiotensin II- or pressure overload-induced cardiomyocyte hypertrophy through calcineurin accumulation.

The gene ankyrin repeat domain 1 (Ankrd1) is an enigmatic gene and may exert pleiotropic function dependent on its expression level, subcellular localization and even types of pathological stress, but it remains unclear how these factors influence the fate of cardiomyocytes. Here we attempted to investigate the role of CARP on cardiomyocyte hypertrophy. In neonatal rat ventricular cardiomyocytes (NRVCs), angiotensin II (Ang II) increased the expression of both calpain 1 and CARP, and also induced cytosolic translocation of CARP, which was abrogated by a calpain inhibitor. In the presence of Ang-II in NRVCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced myocyte hypertrophy, the upregulation of atrial natriuretic peptide and β-myosin heavy chain genes and calcineurin proteins as well as nuclear translocation of nuclear factor of activated T cells. Cyclosporin A attenuated Ad-Ankrd1-enhanced cardiomyocyte hypertrophy. Intra-myocardial injection of Ad-Ankrd1 in mice with transverse aortic constriction (TAC) markedly increased the cytosolic CARP level, the heart weight/body weight ratio, while short hairpin RNA targeting Ankrd1 inhibited TAC-induced hypertrophy. The expression of calcineurin was also significantly increased in Ad-Ankrd1-infected TAC mice. Olmesartan (an Ang II receptor antagonist) prevented the upregulation of CARP in both Ang II-stimulated NRVCs and hearts with pressure overload. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac remodeling through the enhancement of cytosolic translocation of CARP and upregulation of calcineurin.

Abstract 361: Inactivation of Cardiac Foxo1 by Insulin Signaling Is Required for Cardiac Function and Suppression of β-Myosin Heavy Chain Gene Expression

Heart failure is a leading cause of morbidity and mortality in the USA and is closely associated with diabetes mellitus. The molecular link between diabetes and heart failure is incompletely understood. We recently demonstrated that insulin receptor substrate 1, 2 (IRS1, 2) are key components of insulin signaling and their dysfunction mediates insulin resistance, resulting in metabolic dysregulation and heart failure. Loss of IRS1 and IRS2 is associated with downstream Akt inactivation and in turn activation of the forkhead transcription factor Foxo1. To determine the role of Foxo1 in control of heart failure in insulin resistance and diabetes, we generated mice lacking Foxo1 gene specifically in the heart. Mice lacking both IRS1 and IRS2 in adult hearts exhibited severe heart failure, loss of mitochondria, and a remarkable increase in the β-isoform of myosin heavy chain (β-MHC) gene expression, while deletion of cardiac Foxo1 gene largely prevented the heart failure and the loss of mitochondria, and resulted in a decrease in β-MHC expression. The effect of Foxo1 deficiency on rescuing cardiac dysfunction was also observed in db/db mice and high-fat diet (HFD) mice. Using cultures of primary ventricular cardiomyocytes, we found that Foxo1 interacts with the promoter region of β-MHC and stimulates gene expression, mediating an effect of insulin that suppresses β-MHC expression. Taken together, our study suggests that Foxo1 has important roles in promoting diabetic cardiomyopathy and controls β-MHC expression in development of cardiac dysfunction. Targeting Foxo1 and its regulation will provide novel strategies in preventing metabolic and myocardial dysfunction and influencing MHC plasticity in diabetes mellitus.

Concomitant low-dose doxorubicin treatment and exercise.

Cardiotoxicity is a side effect for cancer patients treated with doxorubicin (DOX). We tested the hypothesis that low-intensity aerobic exercise concomitant with DOX treatment would offset DOX-induced cardiotoxicity while also improving the therapeutic efficacy of DOX on tumor progression. B16F10 melanoma cells (3 × 10(5)) were injected subcutaneously into the scruff of 6- to 8-wk-old male C57BL/6 mice (n = 48). A 4 mg/kg cumulative dose of DOX was administered over 2 wk, and exercise (EX) consisted of treadmill walking (10 m/min, 45 min/day, 5 days/wk, 2 wk). Four experimental groups were tested: 1) sedentary (SED) + vehicle, 2) SED + DOX, 3) EX + vehicle, and 4) EX + DOX. Tumor volume was attenuated in DOX and lowest in EX + DOX. DOX-treated animals had less gain in body weight, reduced heart weights (HW), smaller HW-to-body weight ratios, and shorter tibial lengths by the end of the protocol; and exercise did not reverse the cardiotoxic effects of DOX. Despite decreased left ventricular (LV) mass with DOX, cardiomyocyte cross-sectional area, β-myosin heavy chain gene expression, and whole heart systolic (fractional shortening) and diastolic (E/A ratio) function were similar among groups. DOX also resulted in increased LV fibrosis with lower LV end diastolic volume and stroke volume. Myocardial protein kinase B activity was increased with both DOX and EX treatments, and tuberous sclerosis 2 (TSC2) abundance was reduced with EX. Downstream phosphorylation of TSC2 and mammalian target of rapamycin were similar across groups. We conclude that exercise increases the efficacy of DOX in inhibiting tumor growth without mitigating subclinical DOX-induced cardiotoxicity in a murine model of melanoma.

Creatine Kinase Adenosine Triphosphate and Phosphocreatine Energy Supply in a Single Kindred of Patients With Hypertrophic Cardiomyopathy

A lethal and extensively characterized familial form of hypertrophic cardiomyopathy (HC) is due to a point mutation (Arg403Gln) in the cardiac β-myosin heavy chain gene. Although this is associated with abnormal energy metabolism and progression to heart failure in an animal model, in vivo cardiac energetics have not been characterized in patients with this mutation. Noninvasive phosphorus saturation transfer magnetic resonance spectroscopy was used to measure the adenosine triphosphate supplied by the creatine kinase (CK) reaction and phosphocreatine, the heart's primary energy reserve, in 9 of 10 patients from a single kindred with HC caused by the Arg403GIn mutation and 17 age-matched healthy controls. Systolic and diastolic function was assessed by echocardiography in all 10 patients with HC. The patients with HC had impairment of diastolic function and mild systolic dysfunction, when assessed using global systolic longitudinal strain. Myocardial phosphocreatine was significantly decreased by 24% in patients (7.1 ± 2.3 μmol/g) compared with the controls (9.4 ± 1.2 μmol/g; p = 0.003). The pseudo-first-order CK rate-constant was 26% lower (0.28 ± 0.15 vs 0.38 ± 0.07 s⁻¹, p = 0.035) and the forward CK flux was 44% lower (2.0 ± 1.4 vs 3.6 ± 0.9 μmol/g/s, p = 0.001) than in the controls. The contractile abnormalities did not correlate with the metabolic indexes. In conclusion, myocardial phosphocreatine and CK-ATP delivery are significantly reduced in patients with HC caused by the Arg403Gln mutation, akin to previous results from mice with the same mutation. A lack of a relation between energetic and contractile abnormalities suggests the former result from the sarcomeric mutation and not a late consequence of mechanical dysfunction.

Prenatal ultrasound diagnosis of MYH7 non‐compaction cardiomyopathy

We report on two prenatal ultrasound diagnoses of left ventricular non-compaction cardiomyopathy (LVNC) associated with mutation of the cardiac β-myosin heavy chain gene (MYH7). LVNC is characterized by a trabecular meshwork and deep intertrabecular myocardial recesses communicating with the left ventricular cavity. Clinical features range from non-penetrant disease in adult carriers to heart failure, arrhythmia and thromboembolism. Both cases showed cardiomegaly on prenatal ultrasound examinations, with features indicating non-compaction of the myocardium apparent in the third trimester. Mutations in the MYH7 gene were identified postnatally in each case in both the proband and the father. One infant underwent surgical mitral valvuloplasty and a mechanical valve implant later; in the other, left ventricular function was unimpaired at birth. Cardiac function in both cases remained stable at last follow-up. These cases highlight the importance of prenatal ultrasound diagnosis of LVNC and the need for cardiologic and molecular testing of first-degree relatives who may be unknown carriers of an MYH7 mutation.

Sudden Death in an Infant With Angina, Restrictive Cardiomyopathy, and Coronary Artery Bridging

The patient, a female infant aged 9 months, presented to a community hospital emergency room with fever and was referred for cardiac evaluation because of an abnormal chest radiograph. Her ECG showed biatrial enlargement and inferolateral ST depression. An echocardiogram showed a dilated left atrium and a mildly dilated and thickened left ventricle. Systolic function was preserved but diastolic filling showed restrictive physiology. A clinical diagnosis of restrictive cardiomyopathy (RCM) with associated myocardial hypertrophy was made. The patient underwent a comprehensive metabolic screen, as well as genetic testing (Hypertrophic Cardiomyopathy [HCM] Panel, Laboratory for Molecular Medicine, Cambridge, MA), which revealed a mutation (A1157G) in exon 13 of the β-myosin heavy chain ( MYH7 ) gene, resulting in a nonsynonymous amino acid change from tyrosine to cysteine at position 386. This mutation has not been reported in the literature but had been seen previously by the Laboratory in an infant with de novo HCM. The …