ABSTRACT
Despite advances in therapeutics for heart failure and arrhythmias, a substantial proportion of patients with cardiomyopathy do not respond to interventions, indicating a need to identify novel modifiable myocardial pathobiology. Human genetic variation associated with severe forms of cardiomyopathy and arrhythmias has highlighted the crucial role of alternative splicing in myocardial health and disease, given that it determines which mature RNA transcripts drive the mechanical, structural, signalling and metabolic properties of the heart. In this Review, we discuss how the analysis of cardiac isoform expression has been facilitated by technical advances in multiomics and long-read and single-cell sequencing technologies. The resulting insights into the regulation of alternative splicing - including the identification of cardiac splice regulators as therapeutic targets and the development of a translational pipeline to evaluate splice modulators in human engineered heart tissue, animal models and clinical trials - provide a basis for improved diagnosis and therapy. Finally, we consider how the medical and scientific communities can benefit from facilitated acquisition and interpretation of splicing data towards improved clinical decision-making and patient care.
Subject(s)
Cardiomyopathies , Heart Failure , Animals , Humans , Alternative Splicing , Heart Failure/diagnosis , Heart Failure/genetics , Heart Failure/therapy , Myocardium/metabolism , Cardiomyopathies/metabolism , ProteomicsABSTRACT
To adapt to changing hemodynamic demands, regulatory mechanisms modulate actin-myosin-kinetics by calcium-dependent and -independent mechanisms. We investigate the posttranslational modification of human essential myosin light chain (ELC) and identify NIMA-related kinase 9 (NEK9) to interact with ELC. NEK9 is highly expressed in the heart and the interaction with ELC is calcium-dependent. Silencing of NEK9 results in blunting of calcium-dependent ELC-phosphorylation. CRISPR/Cas9-mediated disruption of NEK9 leads to cardiomyopathy in zebrafish. Binding to ELC is mediated via the protein kinase domain of NEK9. A causal relationship between NEK9 activity and ELC-phosphorylation is demonstrated by genetic sensitizing in-vivo. Finally, we observe significantly upregulated ELC-phosphorylation in dilated cardiomyopathy patients and provide a unique map of human ELC-phosphorylation-sites. In summary, NEK9-mediated ELC-phosphorylation is a calcium-dependent regulatory system mediating cardiac contraction and inotropy.
Subject(s)
Actins , Myosin Light Chains , Humans , Animals , Myosin Light Chains/metabolism , Phosphorylation , Actins/metabolism , Zebrafish/metabolism , Calcium/metabolism , NIMA-Related Kinases/genetics , NIMA-Related Kinases/metabolism , Protein Kinases/metabolismABSTRACT
Recently, circular RNAs (circRNAs) have been extensively studied in animals and plants. circRNAs are generated by backsplicing from the same linear transcripts that are canonically spliced to produce, for example, mature mRNAs. circRNAs exhibit tissue-specific expression and are potentially involved in many diseases, among them cardiovascular diseases. The comprehensive analysis of circRNA expression patterns across larger patient cohorts requires a streamlined and cost-effective workflow designed to meet small input requirements. In this article, we present Lexo-circSeq, a targeted RNA sequencing approach that can profile up to 110 circRNAs and their corresponding linear transcripts in one experiment. We established Lexo-circSeq employing total human heart RNA and show that our protocol can detect depletion of a specific circRNA in hiPSC-derived cardiomyocytes. Finally, Lexo-circSeq was applied to biopsies from patients diagnosed with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Interestingly, our results indicate that circular-to-linear-ratios for circSLC8A1 and circRBM33 are deregulated in cardiomyopathy.
