Nonsense Variant PRDM16-Q187X Causes Impaired Myocardial Development and TGF-ß Signaling Resulting in Noncompaction Cardiomyopathy in Humans and Mice.

BACKGROUND: PRDM16 plays a role in myocardial development through TGF-ß (transforming growth factor-beta) signaling. Recent evidence suggests that loss of PRDM16 expression is associated with cardiomyopathy development in mice, although its role in human cardiomyopathy development is unclear. This study aims to determine the impact of PRDM16 loss-of-function variants on cardiomyopathy in humans. METHODS: Individuals with PRDM16 variants were identified and consented. Induced pluripotent stem cell-derived cardiomyocytes were generated from a proband hosting a Q187X nonsense variant as an in vitro model and underwent proliferative and transcriptional analyses. CRISPR (clustered regularly interspaced short palindromic repeats)-mediated knock-in mouse model hosting the Prdm16Q187X allele was generated and subjected to ECG, histological, and transcriptional analysis. RESULTS: We report 2 probands with loss-of-function PRDM16 variants and pediatric left ventricular noncompaction cardiomyopathy. One proband hosts a PRDM16-Q187X variant with left ventricular noncompaction cardiomyopathy and demonstrated infant-onset heart failure, which was selected for further study. Induced pluripotent stem cell-derived cardiomyocytes prepared from the PRDM16-Q187X proband demonstrated a statistically significant impairment in myocyte proliferation and increased apoptosis associated with transcriptional dysregulation of genes implicated in cardiac maturation, including TGF-ß-associated transcripts. Homozygous Prdm16Q187X/Q187X mice demonstrated an underdeveloped compact myocardium and were embryonically lethal. Heterozygous Prdm16Q187X/WT mice demonstrated significantly smaller ventricular dimensions, heightened fibrosis, and age-dependent loss of TGF-ß expression. Mechanistic studies were undertaken in H9c2 cardiomyoblasts to show that PRDM16 binds TGFB3 promoter and represses its transcription. CONCLUSIONS: Novel loss-of-function PRDM16 variant impairs myocardial development resulting in noncompaction cardiomyopathy in humans and mice associated with altered TGF-ß signaling.

SMYD1a protects the heart from ischemic injury by regulating OPA1-mediated cristae remodeling and supercomplex formation.

SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.

Identification of Two Homozygous Variants in MYBPC3 and SMYD1 Genes Associated with Severe Infantile Cardiomyopathy.

Mutations in cardiac genes are one of the primary causes of infantile cardiomyopathy. In this study, we report the genetic findings of two siblings carrying variations in the MYBPC3 and SMYD1 genes. The first patient is a female proband exhibiting hypertrophic cardiomyopathy (HCM) and biventricular heart failure carrying a truncating homozygous MYBPC3 variant c.1224-52G>A (IVS13-52G>A) and a novel homozygous variant (c.302A>G; p.Asn101Ser) in the SMYD1 gene. The second patient, the proband's sibling, is a male infant diagnosed with hypertrophic cardiomyopathy and carries the same homozygous MYBPC3 variant. While this specific MYBPC3 variant (c.1224-52G>A, IVS13-52G>A) has been previously reported to be associated with adult-onset hypertrophic cardiomyopathy, this is the first report linking it to infantile cardiomyopathy. In addition, this work describes, for the first time, a novel SMYD1 variant (c.302A>G; p.Asn101Ser) that has never been reported. We performed a histopathological evaluation of tissues collected from both probands and show that these variants lead to myofibrillar disarray, reduced and irregular mitochondrial cristae and cardiac fibrosis. Together, these results provide critical insight into the molecular functionality of these genes in human cardiac physiology.

Correction to "Histone H4K20 Trimethylation Is Decreased in Murine Models of Heart Disease".

[This corrects the article DOI: 10.1021/acsomega.2c00984.].

Histone H4K20 Trimethylation Is Decreased in Murine Models of Heart Disease.

Heart disease is the leading cause of death in the developed world, and its comorbidities such as hypertension, diabetes, and heart failure are accompanied by major transcriptomic changes in the heart. During cardiac dysfunction, which leads to heart failure, there are global epigenetic alterations to chromatin that occur concomitantly with morphological changes in the heart in response to acute and chronic stress. These epigenetic alterations include the reversible methylation of lysine residues on histone proteins. Lysine methylations on histones H3K4 and H3K9 were among the first methylated lysine residues identified and have been linked to gene activation and silencing, respectively. However, much less is known regarding other methylated histone residues, including histone H4K20. Trimethylation of histone H4K20 has been shown to repress gene expression; however, this modification has never been examined in the heart. Here, we utilized immunoblotting and mass spectrometry to quantify histone H4K20 trimethylation in three models of cardiac dysfunction. Our results show that lysine methylation at this site is differentially regulated in the cardiomyocyte, leading to increased H4K20 trimethylation during acute hypertrophic stress in cell models and decreased H4K20 trimethylation during sustained ischemic injury and cardiac dysfunction in animal models. In addition, we examined publicly available data sets to analyze enzymes that regulate H4K20 methylation and identified two demethylases (KDM7B and KDM7C) and two methyltransferases (KMT5A and SMYD5) that were all differentially expressed in heart failure patients. This is the first study to examine histone H4K20 trimethylation in the heart and to determine how this post-translational modification is differentially regulated in multiple models of cardiac disease.

Mitochondrial calcium uniporter stabilization preserves energetic homeostasis during Complex I impairment.

Calcium entering mitochondria potently stimulates ATP synthesis. Increases in calcium preserve energy synthesis in cardiomyopathies caused by mitochondrial dysfunction, and occur due to enhanced activity of the mitochondrial calcium uniporter channel. The signaling mechanism that mediates this compensatory increase remains unknown. Here, we find that increases in the uniporter are due to impairment in Complex I of the electron transport chain. In normal physiology, Complex I promotes uniporter degradation via an interaction with the uniporter pore-forming subunit, a process we term Complex I-induced protein turnover. When Complex I dysfunction ensues, contact with the uniporter is inhibited, preventing degradation, and leading to a build-up in functional channels. Preventing uniporter activity leads to early demise in Complex I-deficient animals. Conversely, enhancing uniporter stability rescues survival and function in Complex I deficiency. Taken together, our data identify a fundamental pathway producing compensatory increases in calcium influx during Complex I impairment.

The role of demethylases in cardiac development and disease.

Heart failure is a worldwide health condition that currently has limited noninvasive treatments. Heart disease includes both structural and molecular remodeling of the heart which is driven by alterations in gene expression in the cardiomyocyte. Therefore, understanding the regulatory mechanisms which instigate these changes in gene expression and constitute the foundation for pathological remodeling may be beneficial for developing new treatments for heart disease. These gene expression changes are largely preceded by epigenetic alterations to chromatin, including the post-translational modification of histones such as methylation, which alters chromatin to be more or less accessible for transcription factors or regulatory proteins to bind and modify gene expression. Methylation was once thought to be a permanent mark placed on histone or non-histone targets by methyltransferases, but is now understood to be a reversible process after the discovery of the first demethylase, KDM1A/LSD1. Since this time, it has been shown that demethylases play key roles in embryonic development, in maintaining cellular homeostasis and disease progression. However, the role of demethylases in the fetal and adult heart remains largely unknown. In this review, we have compiled data on the 33 mammalian demethylases that have been identified to date and evaluate their expression in the embryonic and adult heart as well as changes in expression in the failing myocardium using publicly available RNA-sequencing and proteomic datasets. Our analysis detected expression of 14 demethylases in the normal fetal heart, and 5 demethylases in the normal adult heart. Moreover, 8 demethylases displayed differential expression in the diseased human heart compared to healthy hearts. We then examined the literature regarding these demethylases and provide phenotypic information of 13 demethylases that have been functionally interrogated in some way in the heart. Lastly, we describe the 6 arginine and lysine residues on histones which have been shown to be methylated but have no corresponding demethylase identified which removes these methyl marks. Overall, this review highlights our current knowledge on the role of demethylases, their importance in cardiac development and pathophysiology and provides evidence for the use of pharmacological inhibitors to combat disease.

Transcriptional regulation by methyltransferases and their role in the heart: highlighting novel emerging functionality.

Methyltransferases are a superfamily of enzymes that transfer methyl groups to proteins, nucleic acids, and small molecules. Traditionally, these enzymes have been shown to carry out a specific modification (mono-, di-, or trimethylation) on a single, or limited number of, amino acid(s). The largest subgroup of this family, protein methyltransferases, target arginine and lysine side chains of histone molecules to regulate gene expression. Although there is a large number of functional studies that have been performed on individual methyltransferases describing their methylation targets and effects on biological processes, no analyses exist describing the spatial distribution across tissues or their differential expression in the diseased heart. For this review, we performed tissue profiling in protein databases of 199 confirmed or putative methyltransferases to demonstrate the unique tissue-specific expression of these individual proteins. In addition, we examined transcript data sets from human heart failure patients and murine models of heart disease to identify 40 methyltransferases in humans and 15 in mice, which are differentially regulated in the heart, although many have never been functionally interrogated. Lastly, we focused our analysis on the largest subgroup, that of protein methyltransferases, and present a newly emerging phenomenon in which 16 of these enzymes have been shown to play dual roles in regulating transcription by maintaining the ability to both activate and repress transcription through methyltransferase-dependent or -independent mechanisms. Overall, this review highlights a novel paradigm shift in our understanding of the function of histone methyltransferases and correlates their expression in heart disease.

Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart.

Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.

Differential stabilities and sequence-dependent base pair opening dynamics of Watson-Crick base pairs with 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxylcytosine.

5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson-Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5'-CG-3' sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5'-T(8)X(9)G(10)-3' sequence of the DDD, were compared. The presence of 5caC at the X(9) base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A(5):T(8), whereas 5caC did not. At the oxidized base pair G(4):X(9), 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C(3):G(10). No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G(4):X(9); each favored Watson-Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N(4) exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes.

NMR analysis of base-pair opening kinetics in DNA.

Base pairing in nucleic acids plays a crucial role in their structure and function. Differences in the base-pair opening and closing kinetics of individual double-stranded DNA sequences or between chemically modified base pairs provide insight into the recognition of these base pairs by DNA processing enzymes. This unit describes how to quantify the kinetics for localized base pairs by observing changes in the imino proton signals by nuclear magnetic resonance spectroscopy. The determination of all relevant parameters using state-of-the art techniques and NMR instrumentation, including cryoprobes, is discussed.

Site-specific stabilization of DNA by a tethered major groove amine, 7-aminomethyl-7-deaza-2'-deoxyguanosine.

A cationic 7-aminomethyl-7-deaza-2'-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G¹A²G³A4X5C6G7C8T9C¹°T¹¹C¹²)2 (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X5 imino resonance remained sharp at 55 °C. Additionally, two 5'-neighboring base pairs, A4:T9 and G³:C¹°, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A4:T9 base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A4:T9 base pair in the unmodified duplex, which confirmed that the overall fraction of the A4:T9 base pair in the open state of the modified duplex decreased. This was also observed for the G³:C¹° base pair, where αK(op) for the G³:C¹° base pair in the modified duplex was 3.0 × 106 versus 4.1 × 106 for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X5:C8 and C6:G7 base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.

Thermodynamic signature of DNA damage: characterization of DNA with a 5-hydroxy-2'-deoxycytidine·2'-deoxyguanosine base pair.

Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One of the oxidation lesions formed, 5-hydroxy-2'-deoxycytidine, has been shown to miscode by some replicative DNA polymerases but not by error prone polymerases capable of translesion synthesis. The 5-hydroxy-2'-deoxycytidine lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine base to be extrahelical so it can enter into the enzyme's active site where it is excised off the DNA backbone to afford an abasic site. The thermodynamic and nuclear magnetic resonance results presented here describe the effect of a 5-hydroxy-2'-deoxycytidine·2'-deoxyguanosine base pair on the stability of two different DNA duplexes. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2'-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that requires the lesion to be extrahelical.

Characterization of DNA with an 8-oxoguanine modification.

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2'-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2-8 kcal mol(-1) over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG•C is fundamentally different than that found at G•C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG•C pairing and those base pairs that are 5' of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.