John David Mably

Mutations in the AARS2 gene are linked to infantile cardiomyopathy; however, the underlying molecular mechanism remains unknown. Here we report that PCBP1, a poly(rC) binding protein, interacts with the AARS2 transcript to mediate its alternative splicing. Cardiomyocyte-specific deletion of Pcbp1 in mice impairs normal splicing and causes premature termination of Aars2, leading to defects in heart development and postnatal lethality. Similarly, mice with a deletion in Aars2 that mimics a disease-causing splicing lesion display heart developmental abnormalities, reminiscent of those in patients with infantile mitochondrial cardiomyopathy. Mechanistically, loss of Pcbp1 or Aars2 in the heart reduces oxidative phosphorylation, a hallmark of patients with AARS2 mutations. This reduction in mitochondrial-encoded proteome activates mitonuclear communication and the unfolded protein response pathway, thereby inducing a compensatory nuclear-encoded mitochondrial gene program. Our findings provide insights into the PCBP1−AARS2 regulatory axis in mitochondrial cardiomyopathy.

Necroptosis, a form of regulated necrosis, culminates in cell membrane rupture. Our lab and others have discovered that lysosomal membrane permeabilization (LMP) is an early and crucial event in this process, preceding membrane rupture. Rapid LMP releases potent lysosomal enzymes, particularly proteases, into the cytosol, actively promoting cell death. Live-cell imaging provides an invaluable tool for detecting LMP during necroptosis in real-time. Several fluorescent dyes are highly effective: (1) pH-sensitive LysoTracker dyes track changes in lysosomal pH. A decrease in fluorescence signal indicates a loss of the lysosomal pH gradient, a primary sign of lysosomal dysfunction, which may be a precursor or direct consequence of LMP. (2) Fluorescein-labeled dextran beads are internalized and accumulate in lysosomes. Their release into the cytosol signals complete LMP and cargo leakage. Here, we observed a progressive loss of Lysotracker fluorescence, with diffusing Dextran fluorescence into the cytosol after necroptosis induction. Thus, the live-cell imaging methodology enables researchers to precisely track the timing and extent of lysosomal dysfunction, contributing to a more comprehensive understanding of necroptosis mechanisms and illuminating potential therapeutic interventions.

Necroptosis, a form of regulated necrosis, culminates in cell membrane rupture. Our lab and others have discovered that lysosomal membrane permeabilization (LMP) is an early and crucial event in this process, preceding membrane rupture. Rapid LMP releases potent lysosomal enzymes, particularly proteases, into the cytosol, actively promoting cell death. Live-cell imaging provides an invaluable tool for detecting LMP during necroptosis in real-time. Several fluorescent dyes are highly effective: (1) pH-sensitive LysoTracker dyes track changes in lysosomal pH. A decrease in fluorescence signal indicates a loss of the lysosomal pH gradient, a primary sign of lysosomal dysfunction, which may be a precursor or direct consequence of LMP. (2) Fluorescein-labeled dextran beads are internalized and accumulate in lysosomes. Their release into the cytosol signals complete LMP and cargo leakage. Here, we observed a progressive loss of Lysotracker fluorescence, with diffusing Dextran fluorescence into the cytosol after necroptosis induction. Thus, the live-cell imaging methodology enables researchers to precisely track the timing and extent of lysosomal dysfunction, contributing to a more comprehensive understanding of necroptosis mechanisms and illuminating potential therapeutic interventions.

Cardiac remodeling in response to disease or tissue damage severely impairs heart function. Therefore, the description of the molecular mechanisms responsible is essential for the development of effective therapies. Trbp (Tarbp2) is a multifunctional RNA-binding protein that is essential during heart development, but its role in the adult heart and cardiac remodeling remains unknown. We generated inducible conditional knockout mice to delete Trbp from cardiomyocytes in young adults (Trbp-cKOs). While Trbp-cKO mice did not display a detectable phenotype, under stress conditions induced by transverse aortic constriction pressure overload, they rapidly developed severe heart failure; this was associated with maladaptive cardiac remodeling and increased interstitial fibrosis. RNA-sequencing revealed the induction of a fibrotic gene expression network and the TGF-β signaling pathway in Trbp-cKO hearts. In cultured neonatal rat ventricle cardiomyocytes (NRCMs), inhibition of Trbp resulted in an induction of the expression of both Tgfβ2 and Ltbp2; in contrast, Trbp overexpression repressed Tgfβ2 expression. Knockdown of Trbp in NRCMs that were co-cultured with neonatal rat cardiac fibroblasts (NRCFs) resulted in an increase in fibrotic gene expression. However, knockdown of Trbp in NRCMs combined with knockdown of Tgfβ2 in NRCFs using the same co-culture system failed to induce the same change in fibrotic gene expression. These data provide evidence for a critical role for Trbp in regulating cardiac fibrosis during cardiac remodeling mediated by cross-talk between cardiomyocytes and fibroblasts. The link to TGF-β signaling also highlights its importance and reveals a novel approach to intervention by targeting of Trbp.

BACKGROUND: Cardiac hypertrophy is an adaptive response to pressure overload aimed at maintaining cardiac function. However, prolonged hypertrophy significantly increases the risk of maladaptive cardiac remodeling and heart failure. Recent studies have implicated long noncoding RNAs in cardiac hypertrophy and cardiomyopathy, but their significance and mechanism(s) of action are not well understood. METHODS: We measured lincRNA-p21 RNA and H3K27ac levels in the hearts of dilated cardiomyopathy patients. We assessed the functional role of lincRNA-p21 in basal and surgical pressure-overload conditions using loss-of-function mice. Genome-wide transcriptome analysis revealed dysregulated genes and pathways. We labeled proteins in proximity to full-length lincRNA-p21 using a novel BioID2-based system. We immunoprecipitated lincRNA-p21-interacting proteins and performed cell fractionation, ChIP-seq (chromatin immunoprecipitation followed by sequencing), and co-immunoprecipitation to investigate molecular interactions and underlying mechanisms. We used GapmeR antisense oligonucleotides to evaluate the therapeutic potential of lincRNA-p21 inhibition in cardiac hypertrophy and associated heart failure. RESULTS: lincRNA-p21 was induced in mice and humans with cardiomyopathy. Global and cardiac-specific lincRNA-p21 knockout significantly suppressed pressure overload-induced ventricular wall thickening, stress marker elevation, and deterioration of cardiac function. Genome-wide transcriptome analysis and transcriptional network analysis revealed that lincRNA-p21 acts in trans to stimulate the NFAT/MEF2 (nuclear factor of activated T cells/myocyte enhancer factor-2) pathway. Mechanistically, lincRNA-p21 is bound to the scaffold protein KAP1 (KRAB-associated protein-1). lincRNA-p21 cardiac-specific knockout suppressed stress-induced nuclear accumulation of KAP1, and KAP1 knockdown attenuated cardiac hypertrophy and NFAT activation. KAP1 positively regulates pathological hypertrophy by physically interacting with NFATC4 to promote the overactive status of NFAT/MEF2 signaling. GapmeR antisense oligonucleotide depletion of lincRNA-p21 similarly inhibited cardiac hypertrophy and adverse remodeling, highlighting the therapeutic potential of inhibiting lincRNA-p21 . CONCLUSIONS: These findings advance our understanding of the functional significance of stress-induced long noncoding RNA in cardiac hypertrophy and demonstrate the potential of lincRNA-p21 as a novel therapeutic target for cardiac hypertrophy and subsequent heart failure.

One of the features of pathological cardiac hypertrophy is enhanced translation and protein synthesis. Translational inhibition has been shown to be an effective means of treating cardiac hypertrophy, although system-wide side effects are common. Regulators of translation, such as cardiac-specific long noncoding RNAs (lncRNAs), could provide new, more targeted therapeutic approaches to inhibit cardiac hypertrophy. Therefore, we generated mice lacking a previously identified lncRNA named CARDINAL to examine its cardiac function. We demonstrate that CARDINAL is a cardiac-specific, ribosome-associated lncRNA and show that its expression was induced in the heart upon pathological cardiac hypertrophy and that its deletion in mice exacerbated stress-induced cardiac hypertrophy and augmented protein translation. In contrast, overexpression of CARDINAL attenuated cardiac hypertrophy in vivo and in vitro and suppressed hypertrophy-induced protein translation. Mechanistically, CARDINAL interacted with developmentally regulated GTP-binding protein 1 (DRG1) and blocked its interaction with DRG family regulatory protein 1 (DFRP1); as a result, DRG1 was downregulated, thereby modulating the rate of protein translation in the heart in response to stress. This study provides evidence for the therapeutic potential of targeting cardiac-specific lncRNAs to suppress disease-induced translational changes and to treat cardiac hypertrophy and heart failure.

BACKGROUND: The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation. METHODS: We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed. RESULTS: We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5 ^+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5 ^+/- /Atf4 ^+/- ), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell–derived cardiomyocytes. CONCLUSIONS: These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.

The surge in reports describing non-coding RNAs (ncRNAs) has focused attention on their possible biological roles and effects on development and disease. ncRNAs have been touted as previously uncharacterized regulators of gene expression and cellular processes, possibly working to fine-tune these functions. The sheer number of ncRNAs identified has outpaced the capacity to characterize each molecule thoroughly and to reliably establish its clinical relevance; it has, nonetheless, created excitement about their potential as molecular targets for novel therapeutic approaches to treat human disease. In this Review, we focus on one category of ncRNAs — long non-coding RNAs — and their expression, functions and molecular mechanisms in cardiac hypertrophy and heart failure. We further discuss the prospects for this specific class of ncRNAs as novel targets for the diagnosis and treatment of these conditions.

SUMMARY: Alanyl-transfer RNA synthetase 2 (AARS2) is a nuclear encoded mitochondrial tRNA synthetase that is responsible for charging of tRNA-Ala with alanine during mitochondrial translation. Homozygous or compound heterozygous mutations in the Aars2 gene, including those affecting its splicing, are linked to infantile cardiomyopathy in humans. However, how Aars2 regulates heart development, and the underlying molecular mechanism of heart disease remains unknown. Here, we found that poly(rC) binding protein 1 (PCBP1) interacts with the Aars2 transcript to mediate its alternative splicing and is critical for the expression and function of Aars2. Cardiomyocyte-specific deletion of Pcbp1 in mice resulted in defects in heart development that are reminiscent of human congenital cardiac defects, including noncompaction cardiomyopathy and a disruption of the cardiomyocyte maturation trajectory. Loss of Pcbp1 led to an aberrant alternative splicing and a premature termination of Aars2 in cardiomyocytes. Additionally, Aars2 mutant mice with exon-16 skipping recapitulated heart developmental defects observed in Pcbp1 mutant mice. Mechanistically, we found dysregulated gene and protein expression of the oxidative phosphorylation pathway in both Pcbp1 and Aars2 mutant hearts; these date provide further evidence that the infantile hypertrophic cardiomyopathy associated with the disorder oxidative phosphorylation defect type 8 (COXPD8) is mediated by Aars2. Our study therefore identifies Pcbp1 and Aars2 as critical regulators of heart development and provides important molecular insights into the role of disruptions in metabolism on congenital heart defects.