Ischaemic heart disease (IHD) is the leading cause of death worldwide. Although myocardial cell death plays a significant role in myocardial infarction (MI), its underlying mechanism remains to be elucidated. To understand the progression of MI and identify potential therapeutic targets, we performed tandem mass tag (TMT)-based quantitative proteomic analysis using an MI mouse model. Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) revealed that the glutathione metabolic pathway and reactive oxygen species (ROS) pathway were significantly downregulated during MI. In particular, glutathione peroxidase 4 (GPX4), which protects cells from ferroptosis (an iron-dependent programme of regulated necrosis), was downregulated in the early and middle stages of MI. RNA-seq and qRT-PCR analyses suggested that GPX4 downregulation occurred at the transcriptional level. Depletion or inhibition of GPX4 using specific siRNA or the chemical inhibitor RSL3, respectively, resulted in the accumulation of lipid peroxide, leading to cell death by ferroptosis in H9c2 cardiomyoblasts. Although neonatal rat ventricular myocytes (NRVMs) were less sensitive to GPX4 inhibition than H9c2 cells, NRVMs rapidly underwent ferroptosis in response to GPX4 inhibition under cysteine deprivation. Our study suggests that downregulation of GPX4 during MI contributes to ferroptotic cell death in cardiomyocytes upon metabolic stress such as cysteine deprivation.

Although both physiological hypertrophy (PHH) and pathological hypertrophy (PAH) of the heart have similar morphological appearances, only PAH leads to fatal heart failure. In the present study, we used RNA sequencing (RNA-Seq) to determine the transcriptomic signatures for both PHH and PAH. Approximately 13–20 million reads were obtained for both models, among which PAH showed more differentially expressed genes (DEGs) (2,041) than PHH (245). The expression of 417 genes was barely detectable in the normal heart but was suddenly activated in PAH. Among them, Foxm1 and Plk1 are of particular interest, since Ingenuity Pathway Analysis (IPA) using DEGs and upstream motif analysis showed that they are essential hub proteins that regulate the expression of downstream proteins associated with PAH. Meanwhile, 52 genes related to collagen, chemokines, and actin showed opposite expression patterns between PHH and PAH. MAZ-binding motifs were enriched in the upstream region of the participating genes. Alternative splicing (AS) of exon variants was also examined using RNA-Seq data for PAH and PHH. We found 317 and 196 exon inclusions and exon exclusions, respectively, for PAH, and 242 and 172 exon inclusions and exclusions, respectively for PHH. The AS pattern was mostly related to gains or losses of domains, changes in activity, and localization of the encoded proteins. The splicing variants of 8 genes (i.e., Fhl1, Rcan1, Ndrg2, Synpo, Ttll1, Cxxc5, Egfl7, and Tmpo) were experimentally confirmed. Multilateral pathway analysis showed that the patterns of quantitative (DEG) and qualitative (AS) changes differ depending on the type of pathway in PAH and PHH. One of the most significant changes in PHH is the severe downregulation of autoimmune pathways accompanied by significant AS. These findings revealed the unique transcriptomic signatures of PAH and PHH and also provided a more comprehensive understanding at both the quantitative and qualitative levels.

The structural proximity and functional coupling between the SR (sarcoplasmic reticulum) and mitochondria have been suggested to occur in the heart. However, the molecular architecture involved in the SR–mitochondrial coupling remains unclear. In the present study, we performed various genetic and Ca2+-probing studies to resolve the proteins involved in the coupling process. By using the bacterial 2-hybrid, glutathione transferase pull-down, co-immunoprecipitation and immunocytochemistry assays, we found that RyR2 (ryanodine receptor type 2), which is physically associated with VDAC2 (voltage-dependent anion channel 2), was co-localized in SR–mitochondrial junctions. Furthermore, a fractionation study revealed that VDAC2 was co-localized with RyR2 only in the subsarcolemmal region. VDAC2 knockdown by targeted short hairpin RNA led to an increased diastolic [Ca2+] (calcium concentration) and abolishment of mitochondrial Ca2+ uptake. Collectively, the present study suggests that the coupling of VDAC2 with RyR2 is essential for Ca2+ transfer from the SR to mitochondria in the heart.

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti‐/pro‐apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N‐terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake‐1 and mitochondrial calcium uptake‐2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca²⁺ uptake in a stable MCU knockdown HeLa cell line and exerted dominant‐negative effects in the wild‐type MCU‐expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation. Synopsis This study reports the crystal structures of the N‐terminal domain (NTD) of wild‐type and a S92A mutant form of MCU. Functional analyses suggest that the N‐terminal domain of MCU is essential for the modulation of MCU activity. Crystal structures of the NTD of WT and a S92A mutant form of MCU were determined. The structure of MCU NTD contains a novel fold important for post‐translational modifications. Functional analysis suggests that MCU NTD is essential for the modulation of MCU activity.

Progressive cardiac hypertrophy owing to pathological stimuli, such as pressure overload, is frequently associated with the development of heart failure, a major cause of morbidity and mortality worldwide. Growing evidence has shown that miRNAs are extensively involved in the pathogenesis of cardiac hypertrophy. In the present study, we examined the hypothesis that the miR-19a/b family acts as a key regulator of cardiac hypertrophy and apoptosis. Forced overexpression of miR-19a/b was sufficient to induce hypertrophy in rat neonatal cardiomyocytes. Luciferase assays revealed that miR-19a/b directly target the anti-hypertrophic genes atrogin-1 and MuRF-1 (muscle RING-finger protein-1). The endogenous expressions of the target genes were down-regulated by miR-19a/b. Pro-hypertrophic calcineurin/NFAT (nuclear factor of activated T-cells) signalling was elevated markedly in the presence of miR-19b, and the calcineurin inhibitor CsA (cyclosporin A) and the PKC (protein kinase C) inhibitor GF10923X significantly attenuated the miR-19b-mediated increase in cell size and expression of hypertrophic markers. Furthermore, miR-19b led to increased cell survival through up-regulation of the NFAT target gene encoding α-crystallin-B and repression of the pro-apoptotic gene Bim (Bcl-2-interacting mediator of cell death) under ER (endoplasmic reticulum) stress conditions. Taken together, the results of the present study demonstrate that the miR-19a/b family regulates phenotypes of cardiomyocytes via suppression of multiple direct target genes.

How cell fate (survival or death) is determined and whether such determination depends on the strength of stimulation has remained unclear. In this study, we discover that the cell fate of cardiomyocytes switches from survival to death with the increase of β-adrenergic receptor (β-AR) stimulation. Mathematical simulations combined with biochemical experimentation of β-AR signalling pathways show that the gradual increment of isoproterenol (a non-selective β₁/β₂-AR agonist) induces the switching response of Bcl-2 expression from the initial increase followed by a decrease below its basal level. The ERK1/2 and ICER-mediated feed-forward loop is the hidden design principle underlying such cell fate switching characteristics. Moreover, we find that β1-blocker treatment increases the survival effect of β-AR stimuli through the regulation of Bcl-2 expression leading to the resistance to cell death, providing new insight into the mechanism of therapeutic effects. Our systems analysis further suggests a novel potential therapeutic strategy for heart disease.