Severe Dilated Cardiomyopathy Research Articles

Systemic primary carnitine deficiency induces severe arrhythmias due to shortening of the QT interval

Systemic primary carnitine deficiency is a rare genetic disease characterized by severe hypokinetic dilated cardiomyopathy and/or severe rhythm disorders. Without carnitine supplementation, evolution is usually towards a fatal cardiac decompensation. While the cardiomyopathy is most likely secondary to the energy deficiency, the mechanism of the rhythm disturbance is unclear, and may be related to a significant shortening of the QT interval. The aim of this work was to describe rhythmic manifestations at the diagnosis of primary carnitine deficiency and their evolution with carnitine supplementation. We conducted a retrospective multicenter study, including all French patients with a confirmed diagnosis of primary carnitine deficiency. Clinical and paraclinical data at diagnosis and during follow-up were collected. QT interval were measured by two pediatric cardiologists according to international recommendations and corrected with the Bazett formula (QTc). Nineteen patients (median age at diagnosis 2.3 years) followed in 8 French centers were included. At diagnosis, 22% of the patients (4/18) had arrhythmia (1 sudden death, 1 ventricular tachycardia and 2 ventricular fibrillations), and 78% (14/18) had a cardiomyopathy. Seven electrocardiograms before initiation of treatment out of the 9 available displayed a QTc < 340 ms indicating the diagnosis of short QT. The mean QTc after carnitine supplementation was 385 ± 24 ms versus 334 ± 21 ms before treatment ( P < 0.01). No patient reached the criteria of short QT syndrome with carnitine supplementation. Three patients died during the survey, which included one sudden death before carnitine supplementation, one non-recoverable rhythmic storm also before initiation of supplementation, and one sudden death in a patient who stopped carnitine supplementation 16 years after diagnosis. Primary carnitine deficiency is associated with a short QT interval that induces severe arrhythmias and sudden death. Carnitine supplementation normalizes the QTc and avoids arrhythmias. The screening of primary carnitine deficiency in neonates would prevent neonatal death.

SR Protein Kinases Regulate the Splicing of Cardiomyopathy-Relevant Genes via Phosphorylation of the RSRSP Stretch in RBM20.

(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead to abnormal gene splicing and have been linked to severe dilated cardiomyopathy (DCM) in human patients and animal models. Interestingly, many of the reported DCM-linked missense mutations in RBM20 are in a highly conserved RSRSP stretch within the RS domain. Recently, it was found that the two Ser residues within this stretch are constitutively phosphorylated, yet the identity of the kinase(s) responsible for phosphorylating these residues, as well as the function of RSRSP phosphorylation, remains unknown. (2) Methods: The ability of three known SR protein kinases (SRPK1, CLK1, and AKT2) to phosphorylate the RBM20 RSRSP stretch and regulate target gene splicing was evaluated by using both in vitro and in vivo approaches. (3) Results: We found that all three kinases phosphorylated S638 and S640 in the RSRSP stretch and regulated RBM20 target gene splicing. While SRPK1 and CLK1 were both capable of directly phosphorylating the RS domain in RBM20, whether AKT2-mediated control of the RS domain phosphorylation is direct or indirect could not be determined. (4) Conclusions: Our results indicate that SR protein kinases regulate the splicing of a cardiomyopathy-relevant gene by modulating phosphorylation of the RSRSP stretch in RBM20. These findings suggest that SR protein kinases may be potential targets for the treatment of RBM20 cardiomyopathy.

Abstract P1106: Loss Of Function Of The Nuclear Envelope Protein LEMD2 Causes DNA Damage-dependent Cardiomyopathy

Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, which primarily affect the heart and skeletal muscle. A mutation in the NEP LEMD2 causes severe cardiomyopathy in humans. However, the roles of LEMD2 in the heart and the pathological mechanisms responsible for its association with cardiac disease are unknown. As a consequence, there is no specific therapy for this cardiomyopathy. To explore the basis of this pathology, we generated mice carrying the human c.T38&gt;G Lemd2 mutation. These mice represent a new preclinical model that phenocopied the human disease and developed severe dilated cardiomyopathy with extensive cardiac fibrosis leading to premature death. At the cellular level, LEMD2 mutant cardiomyocytes exhibited disorganization of the transcriptionally silent heterochromatin associated with the nuclear envelope. Moreover, mice with cardiac-specific deletion of LEMD2 also died shortly after birth due to heart abnormalities. Both LEMD2 mutant models displayed aberrant cardiac gene expression and extensive DNA damage linked to p53 activation. Importantly, cardiomyocyte-specific Lemd2 gene therapy via adeno-associated virus significantly rescued cardiac function in the c.T38&gt;G mice. Together, our results reveal the essentiality of the nuclear envelope protein LEMD2 for genome stability and cardiac function and unveil its mechanistic association with human disease.

Abstract P3030: Fundc1 Mediated Mitochondrial Protein Import As A Novel Mechanism Exacerbating Dilated Cardiomyopathy

Background: Dilated cardiomyopathy (DCM) is a common cause of heart failure and an indication for heart transplantation worldwide. FUN14 Domain Containing 1 (FUNDC1), a canonical mitophagy receptor and mitochondria-associated ER membranes (MAMs) protein, plays an essential role in ischemic heart disease. However, the role of FUNDC1 in DCM remains unidentified. The current study attempted to clarify whether and how FUNDC1 regulates DCM development. Methods: Cardiomyocyte-specific F UNDC 1 gene knockout (CM-FUNDC1 KO) mice were generated to study the impact and potential mechanisms of FUNDC1 in the heart. Mitochondria were isolated from heart tissues, primary mouse neonatal/adult cardiomyocytes, or H9C2 cell lines to probe the role of FUNDC1 in mitochondrial protein import (MPI). Results: All CM-FUNDC1 KO mice developed severe DCM spontaneously and died before 15 months old. The expression of MPI-associated proteins was significantly upregulated in the hearts of CM-FUNDC1 KO mice. Experiments with purified mitochondria demonstrated that MPI efficiency was enhanced by CM-FUNDC1 KO, whereas reduced by FUNDC1 overexpression in an autophagy-independent fashion. We further identified that FUNDC1 selectively downregulated the TIM23 complex without altering other MPI translocases (e.g., TOM20, TIM22 complexes). Downregulation of subunits of TIM23 complex partly reversed the FUNDC1 KO-induced MPI enhancement. Importantly, we demonstrated that FUNDC1 selectively inhibited the import of nuclear gene-encoded respiratory subunits on complex I, IV but not on complex II, III, V or mitochondrial gene-encoded subunits. Finally, FUNDC1 deleted cells exhibited enlarged cell size, retarded cell growth, impaired oxygen consumption rate, and increased mitochondrial superoxide level. All of these phenomena were partly reversed by silence TIM23 complex or reproduced by TIM23 complex overexpression. Conclusions: We demonstrate for the first time that: 1) MPI system plays an important role in the progress of DCM; 2) FUNDC1 is a novel negative regulator of MPI via TIM23 pathway; and 3) FUNDC1 is a promising therapeutic target for DCM.

A biallelic loss-of-function variant in MYZAP is associated with a recessive form of severe dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a primary disorder of the cardiac muscle, characterized by dilatation of the left ventricle and contractile dysfunction. About 50% of DCM cases can be attributed to monogenic causes, whereas the etiology in the remaining patients remains unexplained. We report a family with two brothers affected by severe DCM with onset in the adolescent period. Using exome sequencing, we identified a homozygous premature termination variant in the MYZAP gene in both affected sibs. MYZAP encodes for myocardial zonula adherens protein—a conserved cardiac protein in the intercalated disc structure of cardiomyocytes. The effect of the variant was demonstrated by light and electron microscopy of the heart muscle and immunohistochemical and western blot analysis of the MYZAP protein in the heart tissue of the proband. Functional characterization using patient-derived induced pluripotent stem cell cardiomyocytes revealed significantly lower force and longer time to peak contraction and relaxation consistent with severe contractile dysfunction. We provide independent support for the role of biallelic loss-of-function MYZAP variants in dilated cardiomyopathy. This report extends the spectrum of cardiac disease associated with dysfunction of cardiac intercalated disc junction and sheds light on the mechanisms leading to DCM.

Interrelation between α-Cardiac Actin Treadmilling and Myocardin-Related Transcription Factor-A Nuclear Shuttling in Cardiomyocytes.

Myocardin-related transcription factors (MRTFs) play a central role in the regulation of actin expression and cytoskeletal dynamics that are controlled by Rho GTPases. SRF is a ubiquitous transcription factor strongly expressed in muscular tissues. The depletion of SRF in the adult mouse heart leads to severe dilated cardiomyopathy associated with the down-regulation of target genes encoding sarcomeric proteins including α-cardiac actin. The regulatory triad, composed of SRF, its cofactor MRTFA and actin, plays a major role in the coordination of the nuclear transcriptional response to adapt actin filament dynamics associated with changes in cell shape, and contractile and migratory activities. Most of the knowledge on the regulation of the SRF–MRTF–Actin axis has been obtained in non-muscle cells with α-actin and smooth muscle cells with α-smooth actin. Here, we visualized for the first time by a time-lapse video, the nucleocytoplasmic shuttling of MRTFA induced by serum or pro-hypertrophic agonists such as angiotensin II, phenylephrine and endothelin-1, using an MRTFA-GFP adenovirus in cultures of neonatal rat cardiomyocytes. We showed that an inhibitor of the RhoA/ROCK signaling pathway leads to an α-cardiac actin polymerization disruption and inhibition of MRTFA nucleocytoplasmic shuttling. Moreover, inhibition of the PI3K/Akt signaling pathway also prevents the entry of MRTFA into the nuclei. Our findings point out a central role of the SRF–MRTFA–actin axis in cardiac remodeling.

Aortic valve replacement using On-X valve in a child postorthotopic heart transplantation.

A 20-month-old girl presented with severe dilated cardiomyopathy and decompensated congestive cardiac failure. Despite escalating inotropic and mechanical ventilation support, she required placement on extracorporeal membrane oxygenation and transfer to the transplant centre in Newcastle, England. She was placed on biventricular assist device and then Berlin Heart but failed to show any recovery of ventricular function. She underwent orthotopic heart transplantation at 2 years of age. She developed bacterial endocarditis with Enterococcus faecalis resulting in severe aortic valve regurgitation requiring aortic valve replacement with a 19 mm On-X valve (Airtivion) 11 days after her transplant. Given the size of the donor heart, it was possible to implant a 19-mm valve in this 12 kg child with minimal risk of patient prosthesis mismatch. She was anticoagulated with warfarin (On-X valve INR 2-3 for first 3 months; INR 1.5-2.0 thereafter). Although she suffered several other post-operative complications, including malabsorption, nasojejunal feeding, liver dysfunction, vertebral fractures, renal impairment and renal calcification, and need for repeat opening of her tracheostomy site following her initial decannulation, her aortic valve function has remained stable.

Potential of Medical Management to Mitigate Suction Events in Ventricular Assist Device Patients.

Ventricular suction is a common adverse event in ventricular assist device (VAD) patients and can be due to multiple underlying causes. The aim of this study is to analyze the potential of different therapeutic interventions to mitigate suction events induced by different pathophysiological conditions. To do so, a suction module was embedded in a cardiovascular hybrid (hydraulic-computational) simulator reproducing the entire cardiovascular system. An HVAD system (Medtronic) was connected between a compliant ventricular apex and a simulated aorta. Starting from a patient profile with severe dilated cardiomyopathy, four different pathophysiological conditions leading to suction were simulated: hypovolemia (blood volume: -900 ml), right ventricular failure (contractility -70%), hypotension (systemic vascular resistance: 8.3 Wood Units), and tachycardia (heart rate:185 bpm). Different therapeutic interventions such as volume infusion, ventricular contractility increase, vasoconstriction, heart rate increase, and pump speed reduction were simulated. Their effects were compared in terms of general hemodynamics and suction mitigation. Each intervention elicited a different effect on the hemodynamics for every pathophysiological condition. Pump speed reduction mitigated suction but did not ameliorate the hemodynamics. Administering volume and inducing a systemic vasoconstriction were the most efficient interventions in both improving the hemodynamics and mitigating suction. When simulating volume infusion, the cardiac powers increased, respectively, by 38%, 25%, 42%, and 43% in the case of hypovolemia, right ventricular failure, hypotension, and tachycardia. Finally, a management algorithm is proposed to identify a therapeutic intervention suited for the underlying physiologic condition causing suction.

Dual Specific Phosphatase 7 Exacerbates Dilated Cardiomyopathy, Heart Failure, and Cardiac Death by Inactivating the ERK1/2 Signaling Pathway.

Heart failure is one of the most common but complicated end-stage syndromes in clinical practice. Dilated cardiomyopathy is a myocardial structural abnormality that is associated with heart failure. Dual-specificity phosphatases (DUSPs) are a group of protein phosphatases that regulate signaling pathways in numerous diseases; however, their physiological and pathological impact on cardiovascular disease remains unknown. In the present study, we generated two transgenic mouse models, a DUSP7 knockout and a cardiac-specific DUSP7 overexpressor. Mice overexpressing DUSP7 showed an exacerbated disease phenotype, including severe dilated cardiomyopathy, heart failure, and cardiac death. We further demonstrated that high levels of DUSP7 inhibited ERK1/2 phosphorylation and influenced downstream c-MYC, c-FOS, and c-JUN gene expression but did not affect upstream activators. Taken together, our study reveals a novel molecular mechanism for DUSP7 and provides a new therapeutic target and clinical path to alleviate dilated cardiomyopathy and improve cardiac function.

Prognostic role of myocardial inflammation in patients with undefined left ventricular arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: None. Background Etiology identification and risk stratification represent major issues for patients presenting with undefined left ventricular arrhythmogenic cardiomyopathy (ULVACM). Purpose To investigate the role of genetics and histology for ULVACM classification, management, and risk assessment. Methods We retrospectively analyzed a multicenter cohort of patients (screened n=1037) with ULVACM defined by ventricular arrhythmia (VA) onset, nonischemic late gadolinium enhancement (LGE) limited to the LV, and no severe dilated cardiomyopathy (LVEF≥40%). We selected patients undergoing both next generation sequencing (NGS) screening and endomyocardial biopsy (EMB) for etiology definition. When feasible, immunosuppressive therapy (IST) was used to target active myocardial inflammation (AMI). The study endpoint was a composite of cardiac death, heart transplantation and malignant VAs (VT, VF, appropriate ICD treatment). Results The study cohort is composed by 135 ULVACM patients (age 43±14 years, 63% males, LVEF 55±7 %). NGS identified pathogenic or likely-pathogenic variants (PVs/LPVs) consistent with ACM in 21 cases (16%), whereas EMB showed AMI in 78 patients (58%), including 13/21 PVs/LPVs+ (62%). After reclassification of 86 patients (64%), only 49 (36%) remained ULVACM. IST was started in 41/78 AMI patients (53%), including 9/13 PVs/LPVs+ (69%). Twenty patients (15%) met the study endpoint by 12 months, and 36 (27%) by the end of the study (60±27 months). Beyond malignant VT onset, AMI was the only predictor of events by 12 months (HR 5.0, 95%CI 1.4-18.1, p=0.007). No prognostic role was found for PVs/LPVs, except for the subgroup (n=77) with nonsustained VT onset. Among AMI patients, those treated by IST had a significantly lower occurrence of events, both by 12-months (1/41 vs. 16/37, p&amp;lt;0.001) and later (HR 0.05, 95%CI 0.01-0.21, p&amp;lt;0.001). Results were independently confirmed in PVs/LPVs+ and PVs/LPVs- cases. Excluding the IST population, the association of multiple factors among VT onset, PVs/LPVs, and AMI, resulted in an improved discrimination of arrhythmic risk profiles. Conclusion The combined genetic and histological workup allowed reclassification of up to 64% ULVACM patients, and substantially contributed into prognostic assessment. Furthermore, EMB identified suitable candidates for IST, who showed better outcomes irrespectively of their genotype.

NT‐proBNP as a diagnostic marker of dilated cardiomyopathy in dogs

In dogs of service breeds, cardiopathology is one of the most common diseases of non‐infectious etiology. Dilated cardiomyopathy (DCM) is frequently reported among other heart conditions in large and giant breed dogs. As a result of our previous work, it has been established that DCM of varying severity is recorded in no less than 25% of service dogs. The diagnosis at the later stages of development is not difficult ‐ an enlarged cardiac silhouette on the roentgenogram, enlarged cavities of the heart chambers with echocardiography, ventricular extrasystoles on the electrocardiogram, and a drop in blood pressure during tonometry. Treatment at the late stages of DCM is ineffective and prolongs the dog's lifespan by an average of 3 months. It is advisable to start treatment when the dog is in the early stages of DCM development. However, it is not so easy to identify the disease at the initial stage using visual diagnostics. Therefore, additional methods for diagnosing heart function are required, among which serological analysis for brain natriuretic peptide (NT‐proBNP) can be distinguished. The study aimed to determine the sensitivity of NT‐proBNP in large and giant breed dogs suspected of having DCM disease. Study inclusion criterion: dogs used in security and search work; large or giant breeds; age 2‐8 years; vaccination against rabies, leptospirosis, plague of carnivores, adenovirus infection, parvovirus enteritis, viral hepatitis; absence of diseases of infectious, invasive, and non‐infectious etiology for the last 3 months. In the experiment, 24 dogs were used, which were divided into three groups: group 1 ‐ dogs with significant dilatation of the heart cavities, rhythm disturbances, and a drop in blood pressure (n = 8); Group 2 ‐ dogs with moderate dilatation of the heart cavities, ECG changes, a moderate decrease in blood pressure (n = 8); Group 3 ‐ animals that had no abnormalities during ultrasound diagnostics in the state of the heart (n = 8). An enzyme‐linked immunosorbent assay determined the NT‐proBNP activity. Thus, in dogs of group 1, the NT‐proBNP activity was increased to 4381.7 ± 281.1 pmol / l, which is 20.5 times higher (p &lt;0.05) than in healthy dogs. Severe DCM was confirmed in these dogs. In group 2 animals, the NT‐proBNP level exceeded the values of healthy animals and amounted to 831.7 ± 94.5 pmol / l, which is 3.9 times higher than the normal values. Despite the absence of changes detected during visual diagnostics of the heart, in 2 of 8 dogs of group 3, NT‐proBNP increased to 467.8 ± 15.4 pmol / L. It was increased 2.1 times, which suggested a violation of cardiac activity in these animals. Repeated diagnostics of these 2 dogs 9‐15 months after the start of the study revealed changes characteristic of DCM ‐ changes in the electrical activity of the heart, dilatation of the heart chambers, changes in blood pressure. Based on the experiment results, it was concluded that serological analysis for NT‐proBNP level is a sensitive test that can be included in the list of DCM differential diagnoses.

Antisense Therapy Attenuates Phospholamban p.(Arg14del) Cardiomyopathy in Mice and Reverses Protein Aggregation.

Inherited cardiomyopathy caused by the p.(Arg14del) pathogenic variant of the phospholamban (PLN) gene is characterized by intracardiomyocyte PLN aggregation and can lead to severe dilated cardiomyopathy. We recently reported that pre-emptive depletion of PLN attenuated heart failure (HF) in several cardiomyopathy models. Here, we investigated if administration of a Pln-targeting antisense oligonucleotide (ASO) could halt or reverse disease progression in mice with advanced PLN-R14del cardiomyopathy. To this aim, homozygous PLN-R14del (PLN-R14 Δ/Δ) mice received PLN-ASO injections starting at 5 or 6 weeks of age, in the presence of moderate or severe HF, respectively. Mice were monitored for another 4 months with echocardiographic analyses at several timepoints, after which cardiac tissues were examined for pathological remodeling. We found that vehicle-treated PLN-R14 Δ/Δ mice continued to develop severe HF, and reached a humane endpoint at 8.1 ± 0.5 weeks of age. Both early and late PLN-ASO administration halted further cardiac remodeling and dysfunction shortly after treatment start, resulting in a life span extension to at least 22 weeks of age. Earlier treatment initiation halted disease development sooner, resulting in better heart function and less remodeling at the study endpoint. PLN-ASO treatment almost completely eliminated PLN aggregates, and normalized levels of autophagic proteins. In conclusion, these findings indicate that PLN-ASO therapy may have beneficial outcomes in PLN-R14del cardiomyopathy when administered after disease onset. Although existing tissue damage was not reversed, further cardiomyopathy progression was stopped, and PLN aggregates were resolved.

RBM20S639G mutation is a high genetic risk factor for premature death through RNA-protein condensates

Dilated cardiomyopathy (DCM) is a heritable and genetically heterogenous disease often idiopathic and a leading cause of heart failure with high morbidity and mortality. DCM caused by RNA binding motif protein 20 (RBM20) mutations is diverse and needs a more complete mechanistic understanding. RBM20 mutation S637G (S639G in mice) is linked to severe DCM and early death in human patients. In this study, we generated a RBM20 S639G mutation knock-in (KI) mouse model to validate the function of S639G mutation and examine the underlying mechanisms. KI mice exhibited severe DCM and premature death with a ~ 50% mortality in two months old homozygous (HM) mice. KI mice had enlarged atria and increased ANP and BNP biomarkers. The S639G mutation promoted RBM20 trafficking and ribonucleoprotein (RNP) granules in the sarcoplasm. RNA Seq data revealed differentially expressed and spliced genes were associated with arrhythmia, cardiomyopathy, and sudden death. KI mice also showed a reduction of diastolic stiffness and impaired contractility at both the left ventricular (LV) chamber and cardiomyocyte levels. Our results indicate that the RBM20 S639G mutation leads to RNP granules causing severe heart failure and early death and this finding strengthens the novel concept that RBM20 cardiomyopathy is a RNP granule disease.

Cardiac Decompensation after Dilatation of an Arteriovenous Fistula Stenosis: A Case Report

The arteriovenous fistula (AVF) has been considered for several decades as the preferred approach for hemodialysis because of its longevity and its lower morbidity and mortality rate. However, it can be the cause of several infectious and especially cardiovascular complications. Stenosis is the most common complication of AVF. It is favored by several hemodynamic, cellular and mechanical factors. Reduced dialysis performance and thrombosis are the main risks associated with stenosis. Intervention for dilation is indicated in the presence of any pre-thrombotic stenosis or stenosis that alters the quality of dialysis. However, in patients with heart disease, an assessment of the risk of decompensation is mandatory before considering any gesture of dilation of a fistula. We report a case of acute cardiac decompensation in an 85-year-old patient with severe but stable dilated cardiomyopathy after dilatation of an AVF stenosis. This observation shows that particular attention should be given to elderly subjects with AVF stenosis, especially in the presence of underlying heart disease where closure of the AVF with placement of a tunneled catheter is an alternative that should always be discussed.

Implications of Tropomyosin Phosphorylation in Normal and Cardiomyopathic Hearts

Numerous molecular and biochemical processes regulate protein production in the cell. One of these processes, phosphorylation, allows the cell to rapidly adapt to changing physiological situations. In terminally differentiated cells, such as cardiomyocytes, phosphorylation of sarcomeric proteins controls contraction and relaxation under both normal and stressful conditions. The focus of this review is how phosphorylation of sarcomeric proteins alters physiological performance in cardiac muscle with a particular emphasis on the thin filament protein tropomyosin. This topic is addressed by the examination of tropomyosin isoform expression and its phosphorylation state from embryonic to adult murine development. Next, studies are examined which utilize in vivo model systems to express phosphorylation mimetics and de-phosphorylation genetically-altered tropomyosin transgene constructs. Results show that tropomyosin isoform expression is highly regulated, along with its phosphorylation state. Transgenic mouse hearts which express high levels of a constitutively phosphorylated tropomyosin develop a severe dilated cardiomyopathy and die within a month. A more moderate expression of this phosphorylation mimetic leads to normal systolic performance, but impaired diastolic function. When tropomyosin is dephosphorylated, the transgenic mice develop a compensated cardiac hypertrophy without systolic or diastolic alterations. Interestingly, when dephosphorylated tropomyosin is co-expressed with a hypertrophic cardiomyopathy tropomyosin mutation, the pathological phenotype is rescued with improved cardiac function and no indices of systolic or diastolic dysfunction. These studies demonstrate the functional significance of tropomyosin phosphorylation in determining cardiac performance during both normal and pathological conditions.

Inhibition of Jumonji demethylases reprograms severe dilated cardiomyopathy and prolongs survival

Hypertrophic/dilated cardiomyopathy, often a prequel to heart failure, is accompanied by maladaptive transcriptional changes that contribute to arrythmias and contractile misfunction. Transgenic mice constitutively expressing high levels of calcineurin are known to develop extreme heart hypertrophy, which progresses to dilated cardiomyopathy, and to die several weeks after birth. Here, we characterized aberrant transcriptional and epigenetic pathways in this mouse model and established a pharmacological approach to treat established cardiomyopathy. We found that H3K4me3 (trimethyl histone 3 lysine 4) and H3K9me3 (trimethyl histone 3 lysine 9) Jumonji histone demethylases are markedly increased at the protein level and show enhanced enzymatic activity in diseased hearts. These epigenetic regulators continued to increase with time, further affecting cardiac gene expression. Our findings parallel the lower H3K4me3 and H3K9me3 levels seen in human patients. Inhibition of Jumonji demethylase activities in vivo results in lower histone demethylase enzymatic function in the heart and higher histone methylation levels and leads to partial reduction of heart size, reversal of maladaptive transcriptional programs, improved heart function, and prolonged survival. At the molecular level, target genes of transcription factor myocyte enhancer factor 2 are specifically regulated in response to pharmacological or genetic inhibition of Jumonji demethylases. Similar transcriptional reversal of disease-associated genes is seen in a second disease model based on cardiac mechanical overload. Our findings validate pharmacological inhibitors of Jumonji demethylases as potential therapeutics for the treatment of cardiomyopathies across disease models and provide evidence of the reversal of maladaptive transcriptional reprogramming leading to partial restoration of cardiac function. In addition, this study defines pathways of therapeutic resistance upregulated with disease progression.

Cardiac decompensation and promiscuous prenylation of small GTPases in cardiomyocytes in response to local mevalonate pathway disruption.

Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP-loading and abundance of Ras and Rheb in lipidated protein-enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non-canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 12946: Identification of a LMNA Synonymous Variant Associated With Severe Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is one of the most common cardiac phenotypes caused by mutations of lamin A/C (LMNA) gene in humans. Almost 50% of LMNA variants reported in ClinVar as pathogenic or likely pathogenic are due to frameshift, nonsense, or splicing variants, predictive of haploinsufficiency for LMNA protein. In contrast, synonymous variants that do not immediately suggest a haploinsufficiency mechanism are much harder to interpret. We evaluated a cohort of 57 patients who underwent heart transplant for dilated cardiomyopathy for LMNA variants and identified a novel synonymous variant c.936G&gt;A in the last nucleotide of exon 5 of LMNA in a DCM family. Clinically, LMNA c.936G&gt;A carriers presented with severe DCM, conduction disease and high creatine-kinase levels. Sanger sequencing showed the presence of LMNA c.936G&gt;A in the genomic DNA but not in the cDNA derived from one family member’s heart tissue. RT-PCR using RNA from the patient’s heart tissue was performed with primers that amplified LMNA exons 4-6. DNA fragments amplified by RT-PCR were then cloned into pcDNAII vector and 40 clones were picked and sequenced. An aberrantly spliced mRNA containing the variant and a premature stop codon was identified in 1 of the 40 clones. Real-time quantitative PCR showed significantly lower LMNA mRNA levels in the patient’s heart compared to the controls. Our findings suggest that the c.936G&gt;A LMNA variant resulted in aberrant RNA splicing, which possibly triggers nonsense-mediated RNA decay, leading to reduced mRNA and likely protein expression. These results expand the understanding on the association between synonymous LMNA variants and the molecular pathogenesis of LMNA-DCM.

Abstract 11154: Cardioprotective Effects of Mtss1 Reduction Iin Dilated Cardiomyopathy

Introduction: MTSS1 is an I-BAR protein that interacts with the actin cytoskeleton and the cell membrane and is speculated to play a role in cell motility, tumor metastasis, and cardiogenesis. Enhancer variants that reduce the expression of MTSS1 in the human left ventricle have been associated with cardioprotective traits in genome-wide association studies. However, the effect of MTSS1 downregulation on the development of heart disease has not been studied in experimental models. Here we tested the hypothesis that reduction of MTSS1 protects against dilated cardiomyopathy (DCM) in vitro and in vivo . Methods: We used CRISPR-Cas9 to knock out MTSS1 in induced pluripotent stem cells (iPSCs) harboring pathogenic variants from patients with severe DCM. DCM iPSC lines with and without MTSS1 were then differentiated into cardiomyocytes and phenotyped in vitro . A DCM mouse model containing a TPM1 D230N transgene was crossed with a mouse model heterozygous for Mtss1 knockout. Progeny from this F1 cross were assessed for differences in cardiac structure and function via echocardiography. Results: MTSS1 deletion restored the beta-adrenergic response in DCM iPSC cardiomyocytes. In parallel, Mtss1 deletion partially rescued DCM-associated phenotypes including left ventricular dilation and reduced ejection fraction in mice. Conclusions: Our initial results suggest a causal link between MTSS1 reduction and cardioprotection in DCM. Inhibiting cardiac MTSS1 may hold therapeutic potential for DCM, though the cellular mechanisms through which MTSS1 affects cardiac phenotypes remain under investigation.

Severe dilated cardiomyopathy and ventricular arrhythmia in a patient with Emery‐Dreifuss muscular dystrophy harboring a novel frameshift mutation in EMD

AbstractWe report a case of a 55‐year‐old man with dilated cardiomyopathy and ventricular tachycardia/ventricular fibrillation (VT/VF). He had contracture of neck and ankle flexion from infancy and was found to have dilated cardiomyopathy at the age of 24 years. Intensive cardiac treatment including medication and the use of a defibrillator failed to improve his cardiac function, and heart transplantation was indicated at the age of 55 years. Joint contractures and proximal dominant muscle atrophy with mild weakness suggested Emery‐Dreifuss muscular dystrophy (EDMD). The diagnosis of EDMD1 was made based on genetic analysis of EMD and muscle biopsy showing the absence of emerin. The patient was listed as a candidate for heart transplantation, but he died 3 months later due to sustained VT/VF. The present case emphasizes the necessity of considering timely heart transplantation even when a defibrillator is implanted in patients with EDMD.