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Highlights
• Loss of DNAJC19's DnaJ domain disrupts cardiac mitochondrial structure, leading to abnormal cristae formation in iPSC-CMs.
• Impaired mitochondrial structures lead to an increased mitochondrial respiration, ROS and an elevated membrane potential.
• Mutant iPSC-CMs show sarcomere dysfunction and a trend to more arrhythmias, resembling DCMA-associated cardiomyopathy.
Background
Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy.
Methods
We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca\(^{2+}\) kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tv\(_{HeLa}\)).
Results
Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca\(^{2+}\) concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to β-adrenergic stimulation.
Conclusions
Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca\(^{2+}\) kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.
Ultra-high field cardiac MRI in large animals and humans for translational cardiovascular research
(2023)
A key step in translational cardiovascular research is the use of large animal models to better understand normal and abnormal physiology, to test drugs or interventions, or to perform studies which would be considered unethical in human subjects. Ultrahigh field magnetic resonance imaging (UHF-MRI) at 7 T field strength is becoming increasingly available for imaging of the heart and, when compared to clinically established field strengths, promises better image quality and image information content, more precise functional analysis, potentially new image contrasts, and as all in-vivo imaging techniques, a reduction of the number of animals per study because of the possibility to scan every animal repeatedly. We present here a solution to the dual use problem of whole-body UHF-MRI systems, which are typically installed in clinical environments, to both UHF-MRI in large animals and humans. Moreover, we provide evidence that in such a research infrastructure UHF-MRI, and ideally combined with a standard small-bore UHF-MRI system, can contribute to a variety of spatial scales in translational cardiovascular research: from cardiac organoids, Zebra fish and rodent hearts to large animal models such as pigs and humans. We present pilot data from serial CINE, late gadolinium enhancement, and susceptibility weighted UHF-MRI in a myocardial infarction model over eight weeks. In 14 pigs which were delivered from a breeding facility in a national SARS-CoV-2 hotspot, we found no infection in the incoming pigs. Human scanning using CINE and phase contrast flow measurements provided good image quality of the left and right ventricle. Agreement of functional analysis between CINE and phase contrast MRI was excellent. MRI in arrested hearts or excised vascular tissue for MRI-based histologic imaging, structural imaging of myofiber and vascular smooth muscle cell architecture using high-resolution diffusion tensor imaging, and UHF-MRI for monitoring free radicals as a surrogate for MRI of reactive oxygen species in studies of oxidative stress are demonstrated. We conclude that UHF-MRI has the potential to become an important precision imaging modality in translational cardiovascular research.
Restrictive cardiomyopathy is a rare cardiac disease causing severe diastolic dysfunction, ventricular stiffness and dilated atria. In consequence, it induces heart failure often with preserved ejection fraction and is associated with a high mortality. Since it is a poor clinical prognosis, patients with restrictive cardiomyopathy frequently require heart transplantation. Genetic as well as non-genetic factors contribute to restrictive cardiomyopathy and a significant portion of cases are of unknown etiology. However, the genetic forms of restrictive cardiomyopathy and the involved molecular pathomechanisms are only partially understood. In this review, we summarize the current knowledge about primary genetic restrictive cardiomyopathy and describe its genetic landscape, which might be of interest for geneticists as well as for cardiologists.
Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease caused by heterozygous missense mutations within the gene encoding for the nuclear envelope protein transmembrane protein 43 (TMEM43). The disease is characterized by myocyte loss and fibro-fatty replacement, leading to life-threatening ventricular arrhythmias and sudden cardiac death. However, the role of TMEM43 in the pathogenesis of ACM remains poorly understood. In this study, we generated cardiomyocyte-restricted transgenic zebrafish lines that overexpress eGFP-linked full-length human wild-type (WT) TMEM43 and two genetic variants (c.1073C>T, p.S358L; c.332C>T, p.P111L) using the Tol2-system. Overexpression of WT and p.P111L-mutant TMEM43 was associated with transcriptional activation of the mTOR pathway and ribosome biogenesis, and resulted in enlarged hearts with cardiomyocyte hypertrophy. Intriguingly, mutant p.S358L TMEM43 was found to be unstable and partially redistributed into the cytoplasm in embryonic and adult hearts. Moreover, both TMEM43 variants displayed cardiac morphological defects at juvenile stages and ultrastructural changes within the myocardium, accompanied by dysregulated gene expression profiles in adulthood. Finally, CRISPR/Cas9 mutants demonstrated an age-dependent cardiac phenotype characterized by heart enlargement in adulthood. In conclusion, our findings suggest ultrastructural remodeling and transcriptomic alterations underlying the development of structural and functional cardiac defects in TMEM43-associated cardiomyopathy.
Purpose of Review
Arrhythmogenic cardiomyopathy (ACM) is a genetic disease characterized by life-threatening ventricular arrhythmias and sudden cardiac death (SCD) in apparently healthy young adults. Mutations in genes encoding for cellular junctions can be found in about half of the patients. However, disease onset and severity, risk of arrhythmias, and outcome are highly variable and drug-targeted treatment is currently unavailable.
Recent Findings
This review focuses on advances in clinical risk stratification, genetic etiology, and pathophysiological concepts. The desmosome is the central part of the disease, but other intercalated disc and associated structural proteins not only broaden the genetic spectrum but also provide novel molecular and cellular insights into the pathogenesis of ACM. Signaling pathways and the role of inflammation will be discussed and targets for novel therapeutic approaches outlined.
Summary
Genetic discoveries and experimental-driven preclinical research contributed significantly to the understanding of ACM towards mutation- and pathway-specific personalized medicine.
Arrhythmogenic cardiomyopathy (ACM) is characterized by fibro-fatty replacement of the myocardium, heart failure and life-threatening ventricular arrhythmias. Causal mutations were identified in genes encoding for proteins of the desmosomes, predominantly plakophilin-2 (PKP2) and desmoglein-2 (DSG2). We generated gene-edited knock-out iPSC lines for PKP2 (JMUi001-A-2) and DSG2 (JMUi001-A-3) using the CRISPR/Cas9 system in a healthy control iPSC background (JMUi001A). Stem cell-like morphology, robust expression of pluripotency markers, embryoid body formation and normal karyotypes confirmed the generation of high quality iPSCs to provide a novel isogenic human in vitro model system mimicking ACM when differentiated into cardiomyocytes.
LMNA-related dilated cardiomyopathy is an inherited heart disease caused by mutations in the LMNA gene encoding for lamin A/C. The disease is characterized by left ventricular enlargement and impaired systolic function associated with conduction defects and ventricular arrhythmias. We hypothesized that LMNA-mutated patients' induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) display electrophysiological abnormalities, thus constituting a suitable tool for deciphering the arrhythmogenic mechanisms of the disease, and possibly for developing novel therapeutic modalities. iPSC-CMs were generated from two related patients (father and son) carrying the same E342K mutation in the LMNA gene. Compared to control iPSC-CMs, LMNA-mutated iPSC-CMs exhibited the following electrophysiological abnormalities: (1) decreased spontaneous action potential beat rate and decreased pacemaker current (I\(_f\)) density; (2) prolonged action potential duration and increased L-type Ca\(^{2+}\) current (I\(_{Ca,L}\)) density; (3) delayed afterdepolarizations (DADs), arrhythmias and increased beat rate variability; (4) DADs, arrhythmias and cessation of spontaneous firing in response to β-adrenergic stimulation and rapid pacing. Additionally, compared to healthy control, LMNA-mutated iPSC-CMs displayed nuclear morphological irregularities and gene expression alterations. Notably, KB-R7943, a selective inhibitor of the reverse-mode of the Na\(^+\)/Ca\(^{2+}\) exchanger, blocked the DADs in LMNA-mutated iPSC-CMs. Our findings demonstrate cellular electrophysiological mechanisms underlying the arrhythmias in LMNA-related dilated cardiomyopathy.
About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown and a genetic contribution might be underestimated. Here, we used a next-generation sequencing (NGS) approach and in addition single nucleotide polymor-phism (SNP) arrays for the genetic analysis of two independent index patients without familial medical history. Of note, this genetic strategy revealed a homozygous splice site mutation (DSG2–c.378+1G>T) in the first patient and a nonsense mutation (DSG2–p.L772X) in combination with a large deletion in DSG2 in the second one. In conclusion, a recessive inheritance pattern is likely for both cases, which might contribute to the hidden medical history in both families. This is the first report about these novel loss-of-function mutations in DSG2 that have not been previously identi-fied. Therefore, we suggest performing deep genetic analyses using NGS in combination with SNP arrays also for ACM index patients without obvious familial medical history. In the future, this finding might has relevance for the genetic counseling of similar cases.
Arrhythmogenic cardiomyopathy has been clinically defined since the 1980s and causes right or biventricular cardiomyopathy associated with ventricular arrhythmia. Although it is a rare cardiac disease, it is responsible for a significant proportion of sudden cardiac deaths, especially in athletes. The majority of patients with arrhythmogenic cardiomyopathy carry one or more genetic variants in desmosomal genes. In the 1990s, several knockout mouse models of genes encoding for desmosomal proteins involved in cell–cell adhesion revealed for the first time embryonic lethality due to cardiac defects. Influenced by these initial discoveries in mice, arrhythmogenic cardiomyopathy received an increasing interest in human cardiovascular genetics, leading to the discovery of mutations initially in desmosomal genes and later on in more than 25 different genes. Of note, even in the clinic, routine genetic diagnostics are important for risk prediction of patients and their relatives with arrhythmogenic cardiomyopathy. Based on improvements in genetic animal engineering, different transgenic, knock-in, or cardiac-specific knockout animal models for desmosomal and nondesmosomal proteins have been generated, leading to important discoveries in this field. Here, we present an overview about the existing animal models of arrhythmogenic cardiomyopathy with a focus on the underlying pathomechanism and its importance for understanding of this disease. Prospectively, novel mechanistic insights gained from the whole animal, organ, tissue, cellular, and molecular levels will lead to the development of efficient personalized therapies for treatment of arrhythmogenic cardiomyopathy.