Purpose of Review: Heart failure is a syndrome with poor prognosis and no curative options for the majority of patients. The standard one-size-fits-all-treatment approach, targeting neurohormonal dysregulations, helps to modulate symptoms of heart failure, but fails to address the cause of the problem. Precision medicine aims to go beyond symptom modulation and targets pathophysiological mechanisms that underlie disease. In this review, an overview of how precision medicine can be approached as a treatment strategy for genetic heart disease will be discussed. PLN R14del, a genetic mutation known to cause cardiomyopathy, will be used as an example to describe the potential and pitfalls of precision medicine.Recent Findings: PLN R14del is characterized by several disease hallmarks including calcium dysregulation, metabolic dysfunction, and protein aggregation. The identification of disease-related biological pathways and the effective targeting using several modalities, including gene silencing and signal transduction modulation, may eventually provide novel treatments for genetic heart disease.Summary: We propose a workflow on how to approach precision medicine in heart disease. This workflow focuses on deep phenotyping of patient derived material, including in vitro disease modeling. This will allow identification of therapeutic targets and disease modifiers, to be used for the identification of novel biomarkers and the development of precision medicine approaches for genetic cardiomyopathies.

Members of the fetal-gene-program may act as regulatory components to impede deleterious events occurring with cardiac remodeling, and constitute potential novel therapeutic heart failure (HF) targets. Mitochondrial energy derangements occur both during early fetal development and in patients with HF. Here we aim to elucidate the role of DIO2, a member of the fetal-gene-program, in pluripotent stem cell (PSC)-derived human cardiomyocytes and on mitochondrial dynamics and energetics, specifically. RNA sequencing and pathway enrichment analysis was performed on mouse cardiac tissue at different time points during development, adult age, and ischemia-induced HF. To determine the function of DIO2 in cardiomyocytes, a stable human hPSC-line with a DIO2 knockdown was made using a short harpin sequence. Firstly, we showed the selenoprotein, type II deiodinase (DIO2): the enzyme responsible for the tissue-specific conversion of inactive (T4) into active thyroid hormone (T3), to be a member of the fetal-gene-program. Secondly, silencing DIO2 resulted in an increased reactive oxygen species, impaired activation of the mitochondrial unfolded protein response, severely impaired mitochondrial respiration and reduced cellular viability. Microscopical 3D reconstruction of the mitochondrial network displayed substantial mitochondrial fragmentation. Summarizing, we identified DIO2 to be a member of the fetal-gene-program and as a key regulator of mitochondrial performance in human cardiomyocytes. Our results suggest a key position of human DIO2 as a regulator of mitochondrial function in human cardiomyocytes.

A fundamental goal in the biological sciences is to determine how individual cells with varied gene expression profiles and diverse functional characteristics contribute to development, physiology, and disease. Here, we report a novel strategy to assess gene expression and cell physiology in single living cells. Our approach utilizes fluorescently labeled mRNA-specific anti-sense RNA probes and dsRNA-binding protein to identify the expression of specific genes in real-time at single-cell resolution via FRET. We use this technology to identify distinct myocardial subpopulations expressing the structural proteins myosin heavy chain α and myosin light chain 2a in real-time during early differentiation of human pluripotent stem cells. We combine this live-cell gene expression analysis with detailed physiologic phenotyping to capture the functional evolution of these early myocardial subpopulations during lineage specification and diversification. This live-cell mRNA imaging approach will have wide ranging application wherever heterogeneity plays an important biological role.

RNA-based therapeutics, such as small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs) and messenger RNAs (mRNAs) are promising therapeutics that offer new avenues for targeting molecular pathways underlying heart failure (HF) pathogenesis. This review provides an overview of RNA therapeutics, detailing their mechanisms and potential applications in the treatment of HF. Key pathological processes in HF, including dysregulated calcium handling, myocardial fibrosis, oxidative stress, inflammation and aberrant signalling, are explored to identify how RNA-based therapeutics can be utilized to address these mechanisms. Preclinical studies demonstrating the potential of RNA therapeutics to modulate these pathways are discussed. In addition, the review identifies novel therapeutic targets of HF that may allow more precise and effective interventions, potentially reversing disease progression in HF. In this way, the potential of RNA therapeutics as a next-generation treatment strategy for HF are highlighted, offering hope for more targeted and personalized approaches for HF. Graphical Abstract

Aims: Incomplete penetrance is common in genetic cardiomyopathy, but poorly understood. Here, we investigate the circulating molecular signature in a cohort of patients with one specific phospholamban (PLN) p.Arg14del (R14del, R14Δ/+) pathogenic variant underlying R14Δ/+ cardiomyopathy and its association with disease variability and progression. Methods and results: Targeted proteomics, metabolomics, and lipidomics were performed on plasma from 87 R14Δ/+ carriers across the disease spectrum. Unsupervised clustering of plasma proteomics classified R14Δ/+ carriers into clusters, which were evaluated using clinical data, including heart failure (HF) symptoms, echocardiographic parameters, and clinical follow-up. Metabolomics and lipidomics data were integrated. Five clusters of R14Δ/+ carriers were identified based on plasma proteomics (N = 2612 proteins). Clusters 3, 4, and 5 were enriched for higher N-terminal pro-B-type natriuretic peptide levels, and lower left ventricular ejection fraction, compared with Clusters 1 and 2. Ninety-six out of 148 metabolites were differentially expressed across the clusters. Levels of symmetric dimethylarginine, N-acetyl aspartate, cis-aconitic acid, S-adenosyl-L-methionine, acadesine, and succinate were elevated in disease condition Clusters 3, 4, and 5. Levels of energy metabolism–related metabolites (i.e. adenosine triphosphate and nicotinamide) were elevated in Clusters 1, 3, and 4 and correlated strongly with apoptosis markers, indicating ongoing cardiac damage. Clusters 1 and 2 represent seemingly asymptomatic R14Δ/+ carriers with Cluster 1 suspected at risk for cardiac damage due to elevated apoptosis markers. Cluster 3 shows an intermediate phenotype, and Clusters 4 and 5 consist of R14Δ/+ carriers with end-stage HF. Clinical follow-up confirmed Cluster 1 at risk for R14Δ/+ cardiomyopathy progression due to increased adverse events (HF hospitalization, all-cause mortality, or cardiac device implantation). Conclusion: Molecular profiling of R14Δ/+ carriers reveals subgroups with very distinct risk profiles. Early markers of cardiac damage suggest that stratification may enable timely identification of high-risk individuals and improve understanding of disease variability.

Phospholamban (PLN) p.Arg14del (R14^Δ/+, also known as R14del) is a pathogenic variant that causes inherited cardiomyopathy. RNA therapy improves cardiac function and survival in murine PLN R14^Δ/+. However, the molecular disease mechanisms and potential therapeutic effects of RNA therapy in the human setting remain poorly defined. Proteomic and phosphoproteomic profiling was performed on cardiac tissue from R14^Δ/+ patients (N = 6) and compared to other causes of dilated cardiomyopathy (DCM; N = 10). Findings were validated in CRISPR-Cas9-engineered R14^Δ/+ induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and isogenic controls. To assess reversibility, PLN-targeted RNA therapy using antisense oligonucleotides was applied to iPSC-CMs. Proteomics revealed enrichment of fibrotic pathways, while phosphoproteomics highlighted altered actomyosin structural organization uniquely distinguishing R14^Δ/+ from other DCM. This phosphoproteomic profile was recapitulated in R14^Δ/+ iPSC-CMs. RNA therapy concentration-dependently reduced PLN expression and modified the disease-specific phosphorylation profile. Twenty-eight phosphorylation sites were consistently altered across patient tissue and iPSC-CMs; twenty-two were reversed by RNA therapy and were enriched for cadherin- and actin-binding functions, implicating cytoskeletal remodeling. PLN/LC3 protein aggregates, a hallmark of PLN cardiomyopathy, were reduced after RNA therapy. Functionally, R14^Δ/+ cardiomyocytes exhibited accelerated calcium handling and contractile kinetics, which increased further upon RNA therapy. Human PLN R14^Δ/+ cardiomyopathy is characterized by a distinct phosphoproteomic signature involving cytoskeletal and contractile machinery. PLN-targeted RNA therapy reduced PLN expression, partially normalized these alterations, diminished protein aggregation, and enhanced calcium handling and contractile performance. These findings clarify the molecular mechanisms underlying R14^Δ/+ pathogenesis and support RNA therapy as a promising therapeutic strategy for PLN cardiomyopathy.