Pressure overload induces a transition from cardiac hypertrophy to heart failure, but its underlying mechanisms remain elusive. Here we reconstruct a trajectory of cardiomyocyte remodeling and clarify distinct cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure, by integrating single-cardiomyocyte transcriptome with cell morphology, epigenomic state and heart function. During early hypertrophy, cardiomyocytes activate mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked to ERK1/2 and NRF1/2 transcriptional networks. Persistent overload leads to a bifurcation into adaptive and failing cardiomyocytes, and p53 signaling is specifically activated in late hypertrophy. Cardiomyocyte-specific p53 deletion shows that cardiomyocyte remodeling is initiated by p53-independent mitochondrial activation and morphological hypertrophy, followed by p53-dependent mitochondrial inhibition, morphological elongation, and heart failure gene program activation. Human single-cardiomyocyte analysis validates the conservation of the pathogenic transcriptional signatures. Collectively, cardiomyocyte identity is encoded in transcriptional programs that orchestrate morphological and functional phenotypes.

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are genetically and phenotypically heterogeneous. Cardiac function is improved after treatment in some cardiomyopathy patients, but little is known about genetic predictors of long-term outcomes and myocardial recovery following medical treatment. To elucidate the genetic basis of cardiomyopathy in Japan and the genotypes involved in prognosis and left ventricular reverse remodeling (LVRR), we performed targeted sequencing on 120 DCM (70 sporadic and 50 familial) and 52 HCM (15 sporadic and 37 familial) patients and integrated their genotypes with clinical phenotypes. Among the 120 DCM patients, 20 (16.7%) had TTN truncating variants and 13 (10.8%) had LMNA variants. TTN truncating variants were the major cause of sporadic DCM (21.4% of sporadic cases) as with Caucasians, whereas LMNA variants, which include a novel recurrent LMNA E115M variant, were the most frequent in familial DCM (24.0% of familial cases) unlike Caucasians. Of the 52 HCM patients, MYH7 and MYBPC3 variants were the most common (12 (23.1%) had MYH7 variants and 11 (21.2%) had MYBPC3 variants) as with Caucasians. DCM patients harboring TTN truncating variants had better prognosis than those with LMNA variants. Most patients with TTN truncating variants achieved LVRR, unlike most patients with LMNA variants.

Dilated cardiomyopathy (DCM) is a primary cause of heart failure, life-threatening arrhythmias, and cardiac death. Pathogenic mutations have been identified at the loci of more than 50 genes in approximately 50% of DCM cases, while the etiologies of the remainder have yet to be determined. In this study, we applied whole exome sequencing in combination with segregation analysis to one pedigree with familial DCM, and identified a read-through mutation (c.2459 A > C; p.*820Sext*19) in the myosin light chain kinase 3 gene (MYLK3). We then conducted MYLK3 gene screening of 15 DCM patients (7 familial and 8 sporadic) who were negative for mutation screening of the previously-reported cardiomyopathy-causing genes, and identified another case with a MYLK3 frameshift mutation (c.1879_1885del; p.L627fs*41). In vitro experiments and immunohistochemistry suggested that the MYLK3 mutations identified in this study result in markedly reduced levels of protein expression and myosin light chain 2 phosphorylation. This is the first report that MYLK3 mutations can cause DCM in humans. The clinical phenotypes of DCM patients were consistent with MYLK3 loss-of-function mouse and zebrafish models in which cardiac enlargement and heart failure are observed. Our findings highlight an essential role for cardiac myosin light chain kinase in the human heart.

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A R T I C L E I N F O Keywords:Heart failure Single-cell spatial analysis Immunostaining Single-molecule fluorescence in situ hybridization A B S T R A C T Background: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. Methods and results: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain β (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. Conclusions: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.

Circulation Journal Official Journal of the Japanese Circulation Society http://www. j-circ.or.jp nodular lesions in both lungs on chest computed tomography (CT) were strongly suggestive of sarcoidosis. These findings met the diagnosis of sarcoidosis, the severity of which was evaluated using 18 F-fluoro-deoxyglucose positron emission tomography with CT (FDG-PET/CT) and 123 I-betamethylpiodophenyl-pentadecanoic acid (BMIPP)-201 thallium (TL) single-photon emission CT (SPECT). High FDG uptake was identified in the anterior-to-septal wall of the LV with maximum standard uptake value (SUVmax) of 9.46 ( Figure 1A). The uptake of BMIPP was decreased in the LV anteroseptal wall, which partially corresponded with the higher FDG uptake. Defect scores were 9 and 5 for BMIPP uptake and TL, respectively, according to 17-segment SPECT analysis (Figure 2A). LV ejection fraction (LVEF) determined using ECG-gated software was 45%. The patient underwent cardioverter he physical findings of a 34-year-old woman who presented with palpitations were unremarkable, but electrocardiography (ECG) showed sinus rhythm with complete right bundle branch block ( Figure S1A), and further ECG monitoring indicated premature ventricular contractions and non-sustained ventricular tachycardia. Transthoracic echocardiography showed diffusely impaired left ventricular (LV) function and a thinning ventricular septum. Blood troponin and angiotensin-converting enzyme concentration were normal, as was lysozyme concentration. The patient had recently developed fatigue while walking and ECG indicated complete atrioventricular block (cAVB) with escape rhythm (Figure S1B). On cardiac catheterization there was no coronary artery stenosis, and subsequent endomyocardial biopsy indicated noncaseating epithelioid granuloma ( Figure S2 IMAGES IN CARDIOVASCULAR MEDICINE

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