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.

Cardiovascular aging: from cellular and molecular changes to therapeutic interventions.

Progressive age-induced deterioration in the structure and function of the cardiovascular system involves cardiac hypertrophy, diastolic dysfunction, myocardial fibrosis, arterial stiffness, and endothelial dysfunction. These changes are driven by complex processes that are interconnected, such as oxidative stress, mitochondrial dysfunction, autophagy, inflammation, fibrosis, and telomere dysfunction. In recent years, the advances in research of cardiovascular aging, including the wide use of animal models of cardiovascular aging, elucidated an abundance of cell signaling pathways involved in these processes and brought into sight possible interventions, which span from pharmacological agents, such as metformin, sodium-glucose cotransporter 2-inhibitors, rapamycin, dasatinib and quercetin, to lifestyle changes.

PERM1 regulates energy metabolism in the heart via ERRα/PGC-1α axis.

Aims: PERM1 is a striated muscle-specific regulator of mitochondrial bioenergetics. We previously demonstrated that PERM1 is downregulated in the failing heart and that PERM1 positively regulates metabolic genes known as targets of the transcription factor ERRα and its coactivator PGC-1α in cultured cardiomyocytes. The aims of this study were to determine the effect of loss of PERM1 on cardiac function and energetics using newly generated Perm1-knockout (Perm1 -/-) mice and to investigate the molecular mechanisms of its transcriptional control. Methods and results: Echocardiography showed that ejection fraction and fractional shortening were lower in Perm1 -/- mice than in wild-type mice (both p < 0.05), and the phosphocreatine-to-ATP ratio was decreased in Perm1 -/- hearts (p < 0.05), indicating reduced contractile function and energy reserves of the heart. Integrated proteomic and metabolomic analyses revealed downregulation of oxidative phosphorylation and upregulation of glycolysis and polyol pathways in Perm1 -/- hearts. To examine whether PERM1 regulates energy metabolism through ERRα, we performed co-immunoprecipitation assays, which showed that PERM1 bound to ERRα in cardiomyocytes and the mouse heart. DNA binding and reporter gene assays showed that PERM1 was localized to and activated the ERR target promoters partially through ERRα. Mass spectrometry-based screening in cardiomyocytes identified BAG6 and KANK2 as potential PERM1's binding partners in transcriptional regulation. Mammalian one-hybrid assay, in which PERM1 was fused to Gal4 DNA binding domain, showed that the recruitment of PERM1 to a gene promoter was sufficient to activate transcription, which was blunted by silencing of either PGC-1α, BAG6, or KANK2. Conclusion: This study demonstrates that PERM1 is an essential regulator of cardiac energetics and function and that PERM1 is a novel transcriptional coactivator in the ERRα/PGC-1α axis that functionally interacts with BAG6 and KANK2.

Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome.

Some missense gain-of-function mutations in CACNA1C gene, encoding calcium channel CaV1.2, cause a life-threatening form of long QT syndrome named Timothy syndrome, with currently no clinically-effective therapeutics. Here we report that pharmacological targeting of sigma non-opioid intracellular receptor 1 (SIGMAR1) can restore electrophysiological function in iPSC-derived cardiomyocytes generated from patients with Timothy syndrome and two common forms of long QT syndrome, type 1 (LQTS1) and 2 (LQTS2), caused by missense trafficking mutations in potassium channels. Electrophysiological recordings demonstrate that an FDA-approved cough suppressant, dextromethorphan, can be used as an agonist of SIGMAR1, to shorten the prolonged action potential in Timothy syndrome cardiomyocytes and human cellular models of LQTS1 and LQTS2. When tested in vivo, dextromethorphan also normalized the prolonged QT intervals in Timothy syndrome model mice. Overall, our study demonstrates that SIGMAR1 is a potential therapeutic target for Timothy syndrome and possibly other inherited arrhythmias such as LQTS1 and LQTS2.

YAP mediates compensatory cardiac hypertrophy through aerobic glycolysis in response to pressure overload.

The heart utilizes multiple adaptive mechanisms to maintain pump function. Compensatory cardiac hypertrophy reduces wall stress and oxygen consumption, thereby protecting the heart against acute blood pressure elevation. The nuclear effector of the Hippo pathway, Yes-associated protein 1 (YAP), is activated and mediates compensatory cardiac hypertrophy in response to acute pressure overload (PO). In this study, YAP promoted glycolysis by upregulating glucose transporter 1 (GLUT1), which in turn caused accumulation of intermediates and metabolites of the glycolytic, auxiliary, and anaplerotic pathways during acute PO. Cardiac hypertrophy was inhibited and heart failure was exacerbated in mice with YAP haploinsufficiency in the presence of acute PO. However, normalization of GLUT1 rescued the detrimental phenotype. PO induced the accumulation of glycolytic metabolites, including l-serine, l-aspartate, and malate, in a YAP-dependent manner, thereby promoting cardiac hypertrophy. YAP upregulated the GLUT1 gene through interaction with TEA domain family member 1 (TEAD1) and HIF-1α in cardiomyocytes. Thus, YAP induces compensatory cardiac hypertrophy through activation of the Warburg effect.

Protective mitochondrial fission induced by stress-responsive protein GJA1-20k.

The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.

The lncRNA Caren antagonizes heart failure by inactivating DNA damage response and activating mitochondrial biogenesis.

In the past decade, many long noncoding RNAs (lncRNAs) have been identified and their in vitro functions defined, although in some cases their functions in vivo remain less clear. Moreover, unlike nuclear lncRNAs, the roles of cytoplasmic lncRNAs are less defined. Here, using a gene trapping approach in mouse embryonic stem cells, we identify Caren (short for cardiomyocyte-enriched noncoding transcript), a cytoplasmic lncRNA abundantly expressed in cardiomyocytes. Caren maintains cardiac function under pathological stress by inactivating the ataxia telangiectasia mutated (ATM)-DNA damage response (DDR) pathway and activating mitochondrial bioenergetics. The presence of Caren transcripts does not alter expression of nearby (cis) genes but rather decreases translation of an mRNA transcribed from a distant gene encoding histidine triad nucleotide-binding protein 1 (Hint1), which activates the ATM-DDR pathway and reduces mitochondrial respiratory capacity in cardiomyocytes. Therefore, the cytoplasmic lncRNA Caren functions in cardioprotection by regulating translation of a distant gene and maintaining cardiomyocyte homeostasis.

Nampt Potentiates Antioxidant Defense in Diabetic Cardiomyopathy.

[Figure: see text].

Perm1 regulates cardiac energetics as a downstream target of the histone methyltransferase Smyd1.

The transcriptional regulatory machinery in mitochondrial bioenergetics is complex and is still not completely understood. We previously demonstrated that the histone methyltransferase Smyd1 regulates mitochondrial energetics. Here, we identified Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) as a downstream target of Smyd1 through RNA-seq. Chromatin immunoprecipitation assay showed that Smyd1 directly interacts with the promoter of Perm1 in the mouse heart, and this interaction was significantly reduced in mouse hearts failing due to pressure overload for 4 weeks, where Perm1 was downregulated (24.4 ± 5.9% of sham, p<0.05). Similarly, the Perm1 protein level was significantly decreased in patients with advanced heart failure (55.2 ± 13.1% of donors, p<0.05). Phenylephrine (PE)-induced hypertrophic stress in cardiomyocytes also led to downregulation of Perm1 (55.7 ± 5.7% of control, p<0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I). Pathway enrichment analysis of cardiomyocytes in which Perm1 was knocked-down by siRNA (siPerm1), revealed that the most downregulated pathway was metabolism. Cell stress tests using the Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7% and 23.6% of scrambled-siRNA, respectively, both p<0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known target of ERRα, Ndufv1 (Complex I). Overall, our study demonstrates that Perm1 is an essential regulator of cardiac energetics through ERRα, as part of the Smyd1 regulatory network.

Multiple Levels of PGC-1α Dysregulation in Heart Failure.

Metabolic adaption is crucial for the heart to sustain its contractile activity under various physiological and pathological conditions. At the molecular level, the changes in energy demand impinge on the expression of genes encoding for metabolic enzymes. Among the major components of an intricate transcriptional circuitry, peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) plays a critical role as a metabolic sensor, which is responsible for the fine-tuning of transcriptional responses to a plethora of stimuli. Cumulative evidence suggests that energetic impairment in heart failure is largely attributed to the dysregulation of PGC-1α. In this review, we summarize recent studies revealing how PGC-1α is regulated by a multitude of mechanisms, operating at different regulatory levels, which include epigenetic regulation, the expression of variants, post-transcriptional inhibition, and post-translational modifications. We further discuss how the PGC-1α regulatory cascade can be impaired in the failing heart.

Conduction in the right and left ventricle is differentially regulated by protein kinases and phosphatases: implications for arrhythmogenesis.

The "stress" kinases cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII), phosphorylate the Na+ channel Nav1.5 subunit to regulate its function. However, how the channel regulation translates to ventricular conduction is poorly understood. We hypothesized that the stress kinases positively and differentially regulate conduction in the right (RV) and the left (LV) ventricles. We applied the CaMKII blocker KN93 (2.75 µM), PKA blocker H89 (10 µM), and broad-acting phosphatase blocker calyculin (30 nM) in rabbit hearts paced at a cycle length (CL) of 150-8,000 ms. We used optical mapping to determine the distribution of local conduction delays (inverse of conduction velocity). Control hearts exhibited constant and uniform conduction at all tested CLs. Calyculin (15-min perfusion) accelerated conduction, with greater effect in the RV (by 15.3%) than in the LV (by 4.1%; P < 0.05). In contrast, both KN93 and H89 slowed down conduction in a chamber-, time-, and CL-dependent manner, with the strongest effect in the RV outflow tract (RVOT). Combined KN93 and H89 synergistically promoted conduction slowing in the RV (KN93: 24.7%; H89: 29.9%; and KN93 + H89: 114.2%; P = 0.0016) but not the LV. The progressive depression of RV conduction led to conduction block and reentrant arrhythmias. Protein expression levels of both the CaMKII-δ isoform and the PKA catalytic subunit were higher in the RVOT than in the apical LV (P < 0.05). Thus normal RV conduction requires a proper balance between kinase and phosphatase activity. Dysregulation of this balance due to pharmacological interventions or disease is potentially proarrhythmic. NEW & NOTEWORTHY We show that uniform ventricular conduction requires a precise physiological balance of the activities of calcium/calmodulin-dependent protein kinase II (CaMKII), PKA, and phosphatases, which involves region-specific expression of CaMKII and PKA. Inhibiting CaMKII and/or PKA activity elicits nonuniform conduction depression, with the right ventricle becoming vulnerable to the development of conduction disturbances and ventricular fibrillation/ventricular tachycardia.

Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy.

Obesity induces lipotoxic cardiomyopathy, a condition in which lipid accumulation in cardiomyocytes causes cardiac dysfunction. Here, we show that glycogen synthase kinase-3α (GSK-3α) mediates lipid accumulation in the heart. Fatty acids (FAs) upregulate GSK-3α, which phosphorylates PPARα at Ser280 in the ligand-binding domain (LBD). This modification ligand independently enhances transcription of a subset of PPARα targets, selectively stimulating FA uptake and storage, but not oxidation, thereby promoting lipid accumulation. Constitutively active GSK-3α, but not GSK-3ß, was sufficient to drive PPARα signaling, while cardiac-specific knockdown of GSK-3α, but not GSK-3ß, or replacement of PPARα Ser280 with Ala conferred resistance to lipotoxicity in the heart. Fibrates, PPARα ligands, inhibited phosphorylation of PPARα at Ser280 by inhibiting the interaction of GSK-3α with the LBD of PPARα, thereby reversing lipotoxic cardiomyopathy. These results suggest that GSK-3α promotes lipid anabolism through PPARα-Ser280 phosphorylation, which underlies the development of lipotoxic cardiomyopathy in the context of obesity.

Recruitment of RNA Polymerase II to Metabolic Gene Promoters Is Inhibited in the Failing Heart Possibly Through PGC-1α (Peroxisome Proliferator-Activated Receptor-γ Coactivator-1α) Dysregulation.

BACKGROUND: Proper dynamics of RNA polymerase II, such as promoter recruitment and elongation, are essential for transcription. PGC-1α (peroxisome proliferator-activated receptor [PPAR]-γ coactivator-1α), also termed PPARGC1a, is a transcriptional coactivator that stimulates energy metabolism, and PGC-1α target genes are downregulated in the failing heart. However, whether the dysregulation of polymerase II dynamics occurs in PGC-1α target genes in heart failure has not been defined. METHODS AND RESULTS: Chromatin immunoprecipitation-sequencing revealed that reduced promoter occupancy was a major form of polymerase II dysregulation on PGC-1α target metabolic gene promoters in the pressure-overload-induced heart failure model. PGC-1α-cKO (cardiac-specific PGC-1α knockout) mice showed phenotypic similarity to the pressure-overload-induced heart failure model in wild-type mice, such as contractile dysfunction and downregulation of PGC-1α target genes, even under basal conditions. However, the protein levels of PGC-1α were neither changed in the pressure-overload model nor in human failing hearts. Chromatin immunoprecipitation assays revealed that the promoter occupancy of polymerase II and PGC-1α was consistently reduced both in the pressure-overload model and PGC-1α-cKO mice. In vitro DNA binding assays using an endogenous PGC-1α target gene promoter sequence confirmed that PGC-1α recruits polymerase II to the promoter. CONCLUSIONS: These results suggest that PGC-1α promotes the recruitment of polymerase II to the PGC-1α target gene promoters. Downregulation of PGC-1α target genes in the failing heart is attributed, in part, to a reduction of the PGC-1α occupancy and the polymerase II recruitment to the promoters, which might be a novel mechanism of metabolic perturbations in the failing heart.

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.

Cyclosporine-insensitive mode of cell death after prolonged myocardial ischemia: Evidence for sarcolemmal permeabilization as the pivotal step.

A prominent theory of cell death in myocardial ischemia/reperfusion (I/R) posits that the primary and pivotal step of irreversible cell injury is the opening of the mitochondrial permeability transition (MPT) pore. However, the predominantly positive evidence of protection against infarct afforded by the MPT inhibitor, Cyclosporine A (CsA), in experimental studies is in stark contrast with the overall lack of benefit found in clinical trials of CsA. One reason for the discrepancy might be the fact that relatively short experimental ischemic episodes (<1 hour) do not represent clinically-realistic durations, usually exceeding one hour. Here we tested the hypothesis that MPT is not the primary event of cell death after prolonged (60-80 min) episodes of global ischemia. We used confocal microcopy in Langendorff-perfused rabbit hearts treated with the electromechanical uncoupler, 2,3-Butanedione monoxime (BDM, 20 mM) to allow tracking of MPT and sarcolemmal permeabilization (SP) in individual ventricular myocytes. The time of the steepest drop in fluorescence of mitochondrial membrane potential (ΔΨm)-sensitive dye, TMRM, was used as the time of MPT (TMPT). The time of 20% uptake of the normally cell-impermeable dye, YO-PRO1, was used as the time of SP (TSP). We found that during reperfusion MPT and SP were tightly coupled, with MPT trending slightly ahead of SP (TSP-TMPT = 0.76±1.31 min; p = 0.07). These coupled MPT/SP events occurred in discrete myocytes without crossing cell boundaries. CsA (0.2 µM) did not reduce the infarct size, but separated SP and MPT events, such that detectable SP was significantly ahead of MPT (TSP -TMPT = -1.75±1.28 min, p = 0.006). Mild permeabilization of cells with digitonin (2.5-20 µM) caused coupled MPT/SP events which occurred in discrete myocytes similar to those observed in Control and CsA groups. In contrast, deliberate induction of MPT by titration with H2O2 (200-800 µM), caused propagating waves of MPT which crossed cell boundaries and were uncoupled from SP. Taken together, these findings suggest that after prolonged episodes of ischemia, SP is the primary step in myocyte death, of which MPT is an immediate and unavoidable consequence.

The Smyd Family of Methyltransferases: Role in Cardiac and Skeletal Muscle Physiology and Pathology.

Protein methylation plays a pivotal role in the regulation of various cellular processes including chromatin remodeling and gene expression. SET and MYND domain-containing proteins (Smyd) are a special class of lysine methyltransferases whose catalytic SET domain is split by an MYND domain. The hallmark feature of this family was thought to be the methylation of histone H3 (on lysine 4). However, several studies suggest that the role of the Smyd family is dynamic, targeting unique histone residues associated with both transcriptional activation and repression. Smyd proteins also methylate several non-histone proteins to regulate various cellular processes. Although we are only beginning to understand their specific molecular functions and role in chromatin remodeling, recent studies have advanced our understanding of this relatively uncharacterized family, highlighting their involvement in development, cell growth and differentiation and during disease in various animal models. This review summarizes our current knowledge of the structure, function and methylation targets of the Smyd family and provides a compilation of data emphasizing their prominent role in cardiac and skeletal muscle physiology and pathology.