Cardiac Myosin Heavy Chain Research Articles

The essential role of the miRNA-208a/TARBP2/Sox6 axis in myosin expression in the end stage human heart failure

Abstract Despite diverse aetiologies, failing human hearts (HF) of various origins (coronary artery disease (CAD), dilated cardiomyopathy (DCM), restrictive cardiomyopathy, and hypertrophic cardiomyopathy (HCM)) exhibit impaired contractility and share alterations in MYH6, MYH7, and MYH7B gene expression. Here, we explore the miRNA-208a/TARBP2/Sox6 axis in regulating myosin expression within left ventricles of explanted human hearts. Methods Samples of left ventricles were obtained from explanted hearts of patients with terminal HF (NYHA III-IV) and reduced left ventricle ejection fraction (LVEF <25%) due to CAD (n=10), DCM (n=10), HCM (n=6), and controls (n=5). We analysed the expression of cardiac myosin heavy chains (MYH6, MYH7, MYH7B), cardiac damage markers (ANP, BNP), and heart-related MyoMiRs using quantitative real-time PCR. Results Pre-transplant data for the 31 subjects (25 male, 3 female) included: age 54.4±8.2y, BP 112±18/67±11 mmHg, HR 79±16 min-1, LVEF 27±14%, LVEDD 8.8±15.8mm, RVEDD 32.8±5.1mm, QRS 0.140±0.05s, QT 0.42±0.04s, NT-proBNP 5551±4259 ng/l. In all diseased groups, MYH6 mRNA was downregulated, while MYH7 and MHRT ncRNA were upregulated. We observed strong positive correlations between MYH7, MYH7B, and MHRT transcription, suggesting coordinated expression. The miRNA-208a/miRNA-208b ratio shifted heavily towards miRNA-208b in controls, with a similar effect for miRNA-499 and MYH7B. A strong negative correlation existed between miRNA-499 and MYH7B expression across HF samples. TARBP2 expression decreased slightly in all pathological groups, while Sox6 mRNA increased. Conclusion Elevated Sox6 expression may contribute to MYH6 downregulation and MYH7/MYH7B upregulation. Altered TARBP2 expression could promote cardiomyopathy via Sox6-mediated repression of cardiac slow-twitch myofiber proteins. Changes in miRNA-208a processing leading to Sox6 upregulation may inhibit Sox6-dependent myosin transcript expression.

Pathophysiology of hypereosinophilia-associated heart disease.

Cardiac complications in patients with hypereosinophilia cause significant morbidity and mortality. However, mechanisms of how eosinophilic inflammation causes heart damage are poorly understood. We developed a model of hypereosinophilia-associated heart disease by challenging hypereosinophilic mice with peptide from the cardiac myosin heavy chain. Disease outcomes were measured by histology, immunohistochemistry, flow cytometry, and measurement of cells and biomarkers in peripheral blood. Eosinophil dependence was determined by using eosinophil-deficient mice (ΔdblGATA). Single cells from heart were subjected to single cell RNA sequencing to assess cell composition, subtypes and expression profiles. Mice challenged with myocarditic and control peptide had peripheral blood leukocytosis, but only those challenged with myocarditic peptide had heart inflammation. Heart tissue was infiltrated by eosinophil-rich inflammatory infiltrates associated with cardiomyocyte damage. Disease penetrance and severity were dependent on the presence of eosinophils. Single cell RNA sequencing showed enrichment of myeloid cells, T-cells and granulocytes (neutrophils and eosinophils) in the myocarditic mice. Macrophages were M2 skewed, and eosinophils had an activated phenotype. Gene enrichment analysis identified several pathways potentially involved in pathophysiology of disease. Eosinophils are required for heart damage in hypereosinophilia-associated heart disease. Additionally, myeloid cells, granulocytes and T-cell cooperatively or independently participate in the pathogenesis of hypereosinophilia-associated heart disease.

Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent.

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac β-myosin heavy chain (β-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

Investigation into Cardiac Myhc-α 334-352-Specific TCR Transgenic Mice Reveals a Role for Cytotoxic CD4 T Cells in the Development of Cardiac Autoimmunity.

Myocarditis is one of the major causes of heart failure in children and young adults and can lead to dilated cardiomyopathy. Lymphocytic myocarditis could result from autoreactive CD4+ and CD8+ T cells, but defining antigen specificity in disease pathogenesis is challenging. To address this issue, we generated T cell receptor (TCR) transgenic (Tg) C57BL/6J mice specific to cardiac myosin heavy chain (Myhc)-α 334-352 and found that Myhc-α-specific TCRs were expressed in both CD4+ and CD8+ T cells. To investigate if the phenotype is more pronounced in a myocarditis-susceptible genetic background, we backcrossed with A/J mice. At the fourth generation of backcrossing, we observed that Tg T cells from naïve mice responded to Myhc-α 334-352, as evaluated by proliferation assay and carboxyfluorescein succinimidyl ester staining. The T cell responses included significant production of mainly pro-inflammatory cytokines, namely interferon (IFN)-γ, interleukin-17, and granulocyte macrophage-colony stimulating factor. While the naïve Tg mice had isolated myocardial lesions, immunization with Myhc-α 334-352 led to mild myocarditis, suggesting that further backcrossing to increase the percentage of A/J genome close to 99.99% might show a more severe disease phenotype. Further investigations led us to note that CD4+ T cells displayed the phenotype of cytotoxic T cells (CTLs) akin to those of conventional CD8+ CTLs, as determined by the expression of CD107a, IFN-γ, granzyme B natural killer cell receptor (NKG)2A, NKG2D, cytotoxic and regulatory T cell molecules, and eomesodermin. Taken together, the transgenic system described in this report may be a helpful tool to distinguish the roles of cytotoxic cardiac antigen-specific CD4+ T cells vs. those of CD8+ T cells in the pathogenesis of myocarditis.

Transgenic Mice Expressing Functional TCRs Specific to Cardiac Myhc-α 334-352 on Both CD4 and CD8 T Cells Are Resistant to the Development of Myocarditis on C57BL/6 Genetic Background.

Myocarditis is a predominant cause of congestive heart failure and sudden death in children and young adolescents that can lead to dilated cardiomyopathy. Lymphocytic myocarditis mediated by T cells can result from the recognition of cardiac antigens that may involve CD4 or CD8 T cells or both. In this report, we describe the generation of T cell receptor (TCR) transgenic mice on a C57BL/6 genetic background specific to cardiac myosin heavy chain (Myhc)-α 334-352 and make the following observations: First, we verified that Myhc-α 334-352 was immunogenic in wild-type C57BL/6 mice and induced antigen-specific CD4 T cell responses despite being a poor binder of IAb; however, the immunized animals developed only mild myocarditis. Second, TCRs specific to Myhc-α 334-352 in transgenic mice were expressed in both CD4 and CD8 T cells, suggesting that the expression of epitope-specific TCR is common to both cell types. Third, although T cells from naïve transgenic mice did not respond to Myhc-α 334-352, both CD4 and CD8 T cells from animals immunized with Myhc-α 334-352 responded to the peptide, indicating that antigen priming is necessary to break tolerance. Fourth, although the transgenic T cells could produce significant amounts of interferon-γ and interleukin-17, the immunized animals developed only mild disease, indicating that other soluble factors might be necessary for developing severe myocarditis. Alternatively, the C57BL/6 genetic background might be a major contributing factor for resistance to the development of myocarditis. Taken together, our model permits the determination of the roles of both CD4 and CD8 T cells to understand the disease-resistance mechanisms of myocarditis in a single transgenic system antigen-specifically.

Cooperative mechanisms underlie differences in myocardial contractile dynamics between large and small mammals.

Ca2+ binding to troponin C (TnC) and myosin cross-bridge binding to actin act in a synergistic cooperative manner to modulate myocardial contraction and relaxation. The responsiveness of the myocardial thin filament to the activating effects of Ca2+ and myosin cross-bridge binding has been well-characterized in small mammals (e.g., mice). Given the nearly 10-fold difference in resting heart rates and twitch kinetics between small and large mammals, it is unlikely that the cooperative mechanisms underlying thin filament activation are identical in these two species. To test this idea, we measured the Ca2+ dependencies of steady-state force and the rate constant of force redevelopment (ktr) in murine and porcine permeabilized ventricular myocardium. While murine myocardium exhibited a steep activation-dependence of ktr, the activation-dependent profile of ktr was significantly reduced in porcine ventricular myocardium. Further insight was attained by examining force-pCa and ktr-pCa relationships. In the murine myocardium, the pCa50 for ktr was right-shifted compared with the pCa50 for force, meaning that increases in steady-state force occurred well before increases in the rate of force redevelopment were observed. In the porcine myocardium, we observed a tighter coupling of the force-pCa and ktr-pCa relationships, as evidenced by near-maximal rates of force redevelopment at low levels of Ca2+ activation. These results demonstrate that the molecular mechanisms underlying the cooperative activation of force are a dynamic property of the mammalian heart, involving, at least in part, the species- and tissue-specific expression of cardiac myosin heavy chain isoforms.

Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays

Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5–1.7 µm/s; 25 °C) in the in vitro motility assay as well as maximum actin activated catalytic activity (kcat; 8–9 s−1) and actin concentration for half maximal activity (KATPase; 70–80 µM) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.

Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice

Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies—an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9—to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.

Molecular characterization of a novel MYH7 mutation Q222H in a patient with severe dilated cardiomyopathy.

Mutations in the cardiac myosin heavy chain gene (MYH7) are known to cause both hypertrophic and dilated cardiomyopathy (HCM and DCM), and the use of recombinant human cardiac myosin has enabled the study of the precise biochemical and biophysical effects of these mutations on myosin function at the molecular level. Here we report a case of a patient with severe, early-onset DCM who carries a previously unreported Q222H mutation in MYH7. This mutation affects a residue at the distal end of the ATP binding pocket of cardiac myosin. The Q222H mutant myosin exhibited significantly reduced steady-state actin-activated ATPase activity and a decreased rate of ATP binding in transient kinetic studies. It also showed a ∼50% reduction in actin gliding velocity in an in vitro motility assay. Single-nucleotide turnover assays were performed comparing Q222H mutant myosin 2-headed constructs with either a long S2 tail capable of forming a folded-back auto-inhibited state or a short S2 tail that cannot stably fold back and sequester heads. These studies revealed that the fraction of myosin with a slow ATP hydrolysis rate (super relaxed state, or SRX) thought to represent myosin in an autoinhibited state did not differ significantly between WT and the Q222H mutant myosins. Taken together, the Q222H mutation exerts substantial effects on the motor properties of the cardiac myosin catalytic domain, rather than affecting the availability of myosin heads in the sarcomere. Further transient kinetic assays are underway to delineate precise steps in the actin-myosin ATPase cycle affected by the mutation. Future work also includes comparing these effects with the Q222K mutation that has been reported to cause HCM, to gain further insights into the molecular basis for phenotypic determination of cardiomyopathy.

TBX 5 gene mutation analysis among Tanzanian children with congenital heart diseases using high-resolution melting assays

Early cardiac development is governed by transcription factor genes. TBX5, a T-box transcription factor gene, plays an important role in the development of the second heart field during cardiac septation by promoting cell cycle progression through the enhancement of Cdk6 and hedgehog signaling pathways. TBX5 binds to the promoter region of genes, enhancing the expression of alpha cardiac myosin heavy chain 6 (MYH6), which is a predominant isoform found in human cardiac tissue. TBX5 gene mutations are postulated to cause congenital heart diseases. A casecontrol TBX5 mutational analysis was performed to provide insight into the etiology of sporadic congenital heart diseases in our setting. We used a magnetic induction cycler (mic-PCR), which is a next-generation tool for polymerase chain reaction-high resolution melting assays, to detect mutations in children with sporadic isolated congenital heart diseases. A retrospective casecontrol study was conducted at the Jakaya Kikwete Cardiac Institute. The peripheral blood samples were collected, and DNA was extracted using the Quick-DNA Miniprep Kit. The primers were designed using Primer 3 software, validated using the program BLAST, and checked for hairpin and homo-hetero-dimerization using the IDT oligo analyzer. Real-time polymerase chain reaction (PCR)-high-resolution melting assays for screening TBX5 gene mutations were done using a magnetic induction cycler. We found two (2) TBX5 mutations in exon 5, among patients with Atrial-Ventral Septal Defects (ASVD) and Atrial-Septal Defects (ASD) and none among controls. TBX5 exon 5 is a molecular hotspot for isolated congenital heart diseases.

Abstract 10925: A Small Molecule Inhibitor of Sarcomere Contractility Acutely Relieves Prolonged Relaxation in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases primarily caused by mutations in sarcomeric genes. However, HCM is also associated with mutations in non-sarcomeric proteins and a Finnish founder mutation for HCM in non-sarcomeric protein junctophilin-2 (JPH2) has been identified. MYK-461 is a recently described mechanistically novel small molecule that acts at the sarcomere to specifically inhibit contractility that has been proposed as a treatment for HCM. Here, we use MYK-461 to test whether direct reduction in contractility is sufficient to relieve phenotypic characteristics in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) carrying JPH2 mutation T161K. Hypothesis: Currently all HCM patients are treated the same regardless of the underlying genetic defect. Current medication does not prevent or stop the thickening of the heart muscle; only the symptoms and arrhythmias can be controlled with beta-blockers or calcium blockers. MYK-461 decreases adenosine triphosphatase activity of the cardiac myosin heavy chain to reduce contractility, thus modifying the characteristic pathology of HCM. Methods: Skin fibroblasts from a Finnish patient with HCM causing JPH2 p.(Thr161Lys) were reprogrammed into hiPSCs and further differentiated into CMs. As a control line, an isogenic counterpart was generated using the CRISPR/Cas9 genome editing method. We evaluated the effect of MYK-461 treatment to T161K-hiPSC-CMs by their functional characteristics associated with JPH2 mutation. Results: By video microscopy, we identified increased contractility (BPM; 41±6 SEM, n=9) and prolonged diastolic phase (ms; 394.8±29.7 SEM, n=9) in T161K-hiPSC-CMs. Treatment with MYK-461 reduced contractility (BPM; 36±3 SEM, n=10) and relaxation phase (ms; 293.1±33.6 SEM, n=10) in a dose-dependent manner, displaying functional characteristics closer to isogenic-hiPSC-CMs control group. Conclusions: In conclusion, the results demonstrate that acute reduction in contractility is sufficient to relieve prolonged relaxation. Further, these studies suggest that T161K-hiPSC-CMs provide adequate in vitro platform for modelling the functional effect of MYK-461.

Role of Wnt/β-catenin pathway in cardiac lineage commitment of human umbilical cord mesenchymal stem cells by zebularine and 2'-deoxycytidine

Wnt/β-catenin, a highly conserved signaling pathway, is involved in determining cell fate. During heart development, Wnt signaling controls specification, proliferation and differentiation of cardiac cells. This study is aimed to investigate the role of Wnt/β-catenin signaling in cardiac lineage commitment of human umbilical cord mesenchymal stem cells (hUCMSCs) after treatment with demethylating agents, zebularine and 2'-deoxycytidine (2-DC). hUCMSCs were treated with 20 µM zebularine or 2-DC for 24 h and cultured for 14 days. Control and treated MSCs were analyzed for cardiac lineage commitment at gene and protein levels. Significant upregulation of early and late cardiac markers, GATA4, Nkx2.5, cardiac myosin heavy chain (cMHC), α-actinin, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) was observed in treated MSCs as compared to the untreated control. We also analyzed gene expression of key Wnt/β-catenin signaling molecules in cultures of treated and untreated hUCMSCs at 24 h, and days 3, 7 and 14. The pattern of mRNA gene expression showed that Wnt/β-catenin signaling is regulated during cardiac lineage commitment of hUCMSCs in a time-dependent manner, with the pathway being activated early but inhibited later in cardiac development. Findings of this study can lead us to identify more specific and effective strategies for cardiac lineage commitment.

Na/K‐ATPase Alpha1 Isoform Signaling in Cardiac Metabolism

Understanding cardiac metabolic signaling and its relation to pathological hypertrophy is critical to develop new therapeutic intervention in heart failure (HF). Decreased Na/K‐ATPase (NKA) activity and expression have long been associated with HF in experimental models and in patients. Classically known for its crucial role in transmembrane ion transport, electrical excitability, and contractility, NKA also supports non‐enzymatic receptor functions that differ among major cardiac isoforms (α1 and α2 in rodents). Studies from our group and others have suggested a role of these receptor functions in cardiac myocyte hypertrophy. Using epithelial (LLC‐PK1)‐derived cell lines, we have previously observed a NKA α1‐specific ability to modulate metabolic reserve and capacity. Mechanistically, this property is highly dependent upon NKA α1 ability to regulate the signaling partner Src kinase and is not supported by NKA α2, which lacks the Src binding domain. In this work, our goal was to assess the impact of NKA α1 ablation in the heart and demonstrate the importance of NKA α1 signaling function for proper cardiomyocyte metabolic function. We used a Myh6‐Cre/Lox approach to delete NKA α1 in the mouse heart (cardio α1‐/‐). These animals (3‐month‐old male mice) presented a normal cardiac structure, reduced NKA α1 expression (more than 100‐fold; n = 6‐7; p < 0.01) and increased NKA α2 expression (10‐fold; n = 6‐7; p < 0.01) by Western blot. RNA sequencing followed by gene set enrichment analysis revealed that cardio α1‐/‐ hearts have downregulated pathways related to mitochondrial metabolism, such as cytochrome complex, NADH dehydrogenase complex, respiratory chain, and mitochondrial fatty acid β‐oxidation (n = 3; FDR < 0.05). We next generated human induced pluripotent stem cells (hiPSCs) with a mutant form of the NKA α1 isoform that can pump ions but have blunted Src signaling (A420P mutant) using a CRISPR/Cas9‐mediated knock‐in approach. Genotyping and sequencing confirmed the substitution of alanine to proline in the NaKtide sequence to generate the desired mutation. These cells were differentiated into cardiomyocytes, as confirmed by overexpression of the cardiac markers myosin heavy chain 6 (MYH6) and cardiac muscle troponin T (TNNT2) (more than 10‐fold; n = 3; p < 0.05). Metabolic phenotyping using the Agilent Seahorse Extracellular Flux technology suggested that the A420P mutant hiPSC‐derived cardiomyocytes (A420P iCM) have decreased basal respiration, maximal respiration, spare respiratory capacity, and ATP production compared to the controls (WT iCM) based on changes in oxygen consumption rate (OCR) using the Agilent Seahorse XF Cell Mito Stress Test Kit. In addition, A420P iCM have reduced glycolytic capacity and reserve, based on changes in extracellular acidification rate (ECAR) using the Agilent Seahorse XF Glycolysis Stress Test Kit. Taken together, these results suggest that NKA α1 isoform signaling modulates the cardiomyocyte metabolism. Further studies may reveal a link between NKA/Src signaling, metabolic flexibility, and structural cardiac remodeling in the context of HF and cardiac hypertrophy.

Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell-derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain (MYH7) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin-nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

Clinical characterisation of hypertrophic cardiomyopathy caused by MYH7 gene variants in children

Abstract Background Variants in the cardiac Beta Myosin Heavy chain 7 gene (MYH7) are a common cause of hypertrophic cardiomyopathy (HCM) in adults, but their role in paediatric-onset HCM has not been systematically characterised. This study aims to describe the presentation, clinical characteristics and outcomes of childhood HCM secondary to disease-causing MYH7 variants. Methods Retrospective, longitudinal, data from 70 individuals meeting diagnostic criteria for HCM under the age of 18 years with disease-causing MYH7 variants from a single specialist centre (1991–2019) were collected. A Major Adverse Cardiac Event was defined as sudden cardiac death (SCD), heart failure-related death, cardiac transplantation, haemodynamically-compromising sustained ventricular arrhythmia or appropriate implantable cardioverter defibrillator (ICD) therapy. Results Median age at diagnosis was 9.2 years (IQR 4.2–13.3 years); 47 patients (67.1%) were less than 12 years and 7 (10.0%) were under the age of 1 at diagnosis. Twenty-two patients (31.4%) were probands. MYH7 variants were missense (n=67) or truncating (n=1). Reason for presentation were: family screening (n=45, 64.3%); cardiac symptoms (n=12, 17.1%); incidental finding (n=11, 15.7%); and out of hospital cardiac arrest (n=2, 2.9%). At baseline, mean maximum left ventricular wall thickness (MLVWT) z-score was 9.6 (±5.8), 11 patients (15.7%) had resting left ventricular outflow tract obstruction (left ventricular outflow tract gradient ≥30mmHg). Baseline phenotype did not significantly differ between probands and non-probands (MLVWT Z score 11.9 (±4.5) vs 8.5 (±6.1), p-value 0.0675). Over a median follow up of 3.6 years (IQR 1.8–7.9 years), 10 patients (14.3%) underwent a left ventricular septal myectomy at a median age 6.4 years (IQR 3.4–12.1 years) and 27 (38.6%) had an implantable cardioverter defibrillator (ICD) for primary (n=24, 34.3%) or secondary (n=3, 4.3%) prevention. Three patients (4.3%) died (SCD, n=1; heart-failure related, n=1; non-cardiac, n=1) and 3 (4.3%) underwent cardiac transplantation. Ten patients (14.3%) experienced a MACE. Patients who experienced a MACE were more likely to be probands [n= 6 (60.0%) vs n=16 (26.7%); p=0.036] but did not differ in terms of baseline phenotype (p=0.134). Conclusion MYH7 variants can cause infantile and childhood-onset disease, which is associated with significant early cardiac morbidity and mortality. Adverse outcomes were more common in those presenting as probands. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Medical Research Council, Great Ormond Street Hospital charity.

Organisational alteration of cardiac myofilament proteins by hyperglycaemia in mouse embryonic stem cell-derived cardiomyocytes.

The exposure of the developing foetal heart to hyperglycaemia in mothers with diabetes mellitus is a major risk factor for foetal cardiac complications that lead to heart failure. We studied the effects of hyperglycaemia on the layout of cardiac myofilament proteins in stem cell-derived cardiomyocytes and their possible underlying mechanisms. Mouse embryonic stem cells (mESCs) were differentiated into cardiac-like cells and cultured in media containing baseline- or high glucose concentrations. Cellular biomarkers were detected using Western blot analysis, immunocytochemistry, 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay, and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay. High glucose decreased the proportion of cardiac troponin T and α-actinin 2 positive mESCs as well as disrupted the α-actinin 2 striated pattern and the distribution of the cardiac myosin heavy chain α- and β isoforms. However, there was no alteration of the cellular EdU uptake nor the expression of the receptor of advanced glycation end-product (RAGE). High glucose also increased the presence of the oxidative stress marker nitrotyrosine as well as the number of TUNEL-stained nuclei in cardiac-like cells. Treatment with the antioxidant N-acetyl cysteine decreased the number of TUNEL-stained cells in high glucose and improved the α-actinin 2 striated pattern. Hyperglycaemia negatively impacted the expression and cellular organisation of cardiac myofilament proteins in mESC-derived cardiomyocytes through oxidative stress. The results add further insights into the pathophysiological mechanisms of cardiac contractile dysfunction in diabetic cardiac developmental disease.

FUNDC1 is Essential for Maintaining Cardiac Function During Prolonged Fasting

Mitochondrial quality control via mitophagy is necessary to ensure proper metabolic functioning. However, various heart diseases have been associated with mitochondrial dysfunction and thus linked to increased mortality. Our past studies have shown a time-dependent differential effect of fasting on cardiac mitophagy. Specifically, one-day fasting accelerated mitophagy flux in the mouse heart, whereas two-day fasting actually reduced mitophagy flux, which was associated with the impairment of cardiac function. However, it remains unclear if the reduced mitophagy was responsible for the impaired cardiac function. In the present study, we determined the functional role of mitophagy in the heart during fasting using mice lacking FUNDC1, a mitochondrial outer-membrane protein that serves as a mitophagy receptor. We demonstrated that a two-day fasting impaired cardiac function in wild-type mice, which was exacerbated in FUNDC1 knockout (KO) mice as determined by echocardiography and hemodynamic measurements. This suggests that FUNDC1 or FUNDC1-mediated mitophagy was essential for maintaining cardiac function during fasting. Additionally, the total ATP levels in the hearts showed no dramatic difference between fasted wild type mice and FUNDC1 KO mice, suggesting that the exacerbated cardiac function in FUNDC1 KO mice was likely unrelated to ATP production. Interestingly, FUNDC1 deficiency reduced the expression of cardiac alpha myosin heavy chain (α-MHC) but increased that of β-MHC, a gene that normally expresses during fetal development stage and has the lowest contractile capability among the 3 MHC isoforms. Thus, the altered expression of α-MHC and β-MHC might have contributed to the more severe cardiac dysfunction in FUNDC1 KO mice during fasting.

Levels of anti-B13 antibodies over time in a cohort of chronic infected by Trypanosoma cruzi. Its relationship with specific treatment and clinical status

The immunodominant B13 protein of Trypanosoma cruzi is found on the surface of trypomastigotes and exhibits cross-reactivity with the human cardiac myosin heavy chain; for which antibodies against this parasitic antigen may be involved in the development of disease pathology. In a cohort of chronically T. cruzi-infected adults, undergoing trypanocidal treatment, or not, we, therefore, decided to evaluate the levels of anti-B13 antibodies (ELISA-B13) and its eventual relationship with heart complaints. Two hundred twenty-eight serum samples from 76 chronically infected adults with an average follow-up of 24 years were analyzed. Thirty of them had received trypanocidal treatment. Among treated patients, anti-B13 Ab levels in successive samples showed a significant decrease in reactivity as the years after treatment increased (ANOVA test, p=0.0049). At the end of the follow-up, 36.7% became non-reactive for ELISA B13. Untreated patients did not have significant variations in the level of anti-B13 antibodies during follow-up. None of the treated patients had electrocardiographic changes compatible with chronic chagasic cardiomyopathy, whereas 21.7% of those undergoing no treatment did show such kind of pathological electrocardiogram tracings. ELISA-B13 was reactive in all cases with heart involvement. Among untreated patients, there were no significant differences in anti-B13 antibodies when comparing individuals without proven pathology with those with chronic chagasic cardiomyopathy. Although treatment with trypanocidal drugs was followed by decreased anti-B13 antibody levels, such assessment was unhelpful in differentiating the evolution of chronic chagasic heart disease.

Effects of Cardiac Myosin Heavy Chain Isoform Composition on Contraction Kinetics and Calcium Transients in Adult Rat Cardiomyocytes

Myosin is an essential structural and functional protein of the sarcomere. In rat ventricular myocardium two isoforms of myosin heavy chain (MyHC) are expressed, α-MyHC and β-MyHC. The two MyHC isoforms show different cross bridge kinetics. Strikingly, in the ventricular rat myocardium, cardiomyocytes with different MyHC composition are connected and contract without producing functional disbalances.

Rescue of cardiac function in obese type-2 diabetic mice by transfer of a human longevity gene

Abstract Background Healthy longevity is the result of the interaction between favourable environment and unique genetic makeup. We showed that horizontal transfer of a longevity-associated gene variant (LAV-BPIFB4) improves endothelial function and accelerates the recovery from ischemia. Purpose To determine if the benefit of LAV-BPIFB4 gene therapy can be extended to diabetic cardiomyopathy. Methods and results We confirmed that human diabetic patients with heart failure (n=13) show a decreased cardiac expression of BPIFB4 compared with healthy subjects (n=10). Obese db/db mice received a systemic injection of adeno-associated viral vector (AAV9)-LAV-BPIFB4, AAV9-wild type (WT)-BPIFB4 (both 100 μL at 1×1012 GC/mL) or vehicle before the onset of cardiomyopathy, and were euthanised four weeks later for histological, metabolic and transcriptional analyses. Echocardiographic evaluation (n=8/group), performed at baseline and after gene therapy, showed that LAV-BPIFB4 treatment, despite not resolving hyperglycaemia, improved left ventricular function compared with the other groups. Histological analyses of the hearts (n=5 to 10/group) revealed that LAV-BPIFB4 reduced myocardial fibrosis and increased angiogenesis compared with vehicle and WT-hearts; moreover, LAV increased the expression of the alpha-isoform of the cardiac myosin heavy chain, which is associated with a superior cardiomyocyte contractility. Interestingly, LAV-BPIFB4 treatment induced an increase in cardiac SDF1 expression compared with WT and vehicle, despite the mechanism linking the two events is still unknown. The oral administration of the CXCR4 antagonist AMD-070, given at 2 mg/kg/day for four weeks, abolished several of the beneficial effects exerted by the LAV-BPIFB4 therapy in the obese diabetic mice, as assessed by echocardiography and histology (n=7/group). At the molecular level, next-generation RNA sequencing (n=3 to 4 /group) showed 8 genes were differentially expressed by LAV-BPIFB4-hearts compared with vehicle-hearts. These genes are associated with mitochondrial and metabolic functions. Among them, changes in the UCP3, HMGCS2, CS, ATPB and TOMM20 expression were also validated at the protein level by western blotting. Lipidomics using ultrahigh-performance liquid chromatography-mass spectrometry (n=6 or 7/group) showed 63 metabolites differentially expressed by LAV-BPIFB4- compared with vehicle-hearts, with only 3 (two cardiolipins and one glycerophospholipid) returning close to the non-diabetic phenotype following LAV-BPIFB4 treatment. Conclusions This study newly shows the possibility of transferring the benefit of salutary polymorphic gene variants to protect the cardiovascular system from metabolic pressure. Rather than combating pathogenic mechanisms, the strategy activates alternative pathways overriding disease risk factors. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): British Heart Foundation project grant “Longevity-associated BPIFB4 gene therapy for treatment of ischemic disease”