PERM1 was initially identified as a new downstream target of PGC-1α and ERRs that regulates mitochondrial bioenergetics in skeletal muscle. Subsequently, we and other groups demonstrated that PERM1 is also a positive regulator of mitochondrial bioenergetics in the heart. However, the exact mechanisms of regulatory functions of PERM1 remain poorly understood. O-GlcNAcylation is a post-translational modification of proteins that are regulated by two enzymes: O-GlcNAc transferase (OGT) that adds O-GlcNAc to proteins; O-GlcNAcase (OGA) that removes O-GlcNAc from proteins. O-GlcNAcylation is a powerful signaling mechanism mediating cellular responses to stressors and nutrient availability, which, among other targets, may influence cardiac metabolism. We hypothesized that PERM1 regulates mitochondrial energetics in cardiomyocytes through modulation of O-GlcNAcylation. We found that overexpression of PERM1 decreased the total levels of O-GlcNAcylated proteins, concomitant with decreased OGT and increased OGA expression levels. Luciferase gene reporter assay showed that PERM1 significantly decreases the promoter activity of Ogt without changing the promoter activity of Oga. The downregulation of OGT by PERM1 overexpression was mediated through its interaction with E2F1, a known transcription repressor of Ogt. A deliberate increase of O-GlcNAcylation through Oga silencing in cardiomyocytes decreased the basal and maximal mitochondrial respiration and ATP production rates, all of which were completely restored by PERM1 overexpression. Furthermore, excessive O-GlcNAcylation caused by the loss of PERM1 led to the increase of O-GlcNAcylated PGC-1α, a master regulator of mitochondrial bioenergetics, concurrent with the dissociation of PGC-1α from PPARα, a well-known transcription factor that regulates fatty acid β-oxidation. We conclude that PERM1 positively regulates mitochondrial energetics, in part, via suppressing O-GlcNAcylation in cardiac myocytes.

Reduced muscle contractility and mitochondrial bioenergetics are the hallmarks of systolic heart failure. There is currently no therapy targeting both. Here, we show that gene delivery of Perm1 via adeno-associated virus (AAV) simultaneously enhances cardiac contractility and mitochondrial biogenesis in C57BL6 mice. Moreover, we found that PERM1 interacts with troponin C (TnC), a key contractile protein in striated muscle, and that AAV-Perm1 led to the upregulation of TnC. This study suggests that gene delivery of Perm1 may be a novel therapeutic approach to treat systolic heart failure by simultaneously restoring cardiac contractility and mitochondrial bioenergetics.NEW & NOTEWORTHY Perm1 gene delivered with AAV9 enhances cardiac contractility in mice, and it is concomitant with the increase of mitochondrial bioenergetics and upregulation of TnC. This is the first study showing that PERM1, previously known as a striated muscle-specific mitochondrial regulator, also positively regulates cardiac contractility.

Heart failure remains the leading cause of death in the U.S., and 50% of patients with heart failure still die within 5 years after diagnosis. Mitochondrial impairment and contractile dysfunction are the hallmarks of heart failure with reduced ejection fraction (HFrEF). Yet, there is currently no therapeutic strategy targeting both mitochondria and muscle contractility. PERM1 is a striated-muscle-specific regulator of mitochondrial bioenergetics and is predominantly expressed in the heart and skeletal muscle. Our recent studies demonstrated that PERM1 is downregulated in human and mouse HFrEF hearts and that loss of PERM1 in mice leads to reduced contractility and energy reserve in the heart. However, it is largely unknown whether PERM1 positively regulates both muscle contractility and energetics in the heart. Here, we performed the gene delivery of Perm1 to the heart in C57BL6 wild-type mice (8-12 weeks old) through retro-orbital injection of the adenovirus-associated virus (AAV)9 vector carrying Perm1 (AAV-PERM1). The protein expression levels of PERM1 in the heart was increased by 3 fold as compared with control (mice injected with AAV9-GFP vectors), while there was no change in PERM1 expression in skeletal muscle (n=4/group). Strikingly, AAV-PERM1 mice exhibited a significant increase in ejection fraction (EF) and fractional shortening (FS) (AAV-GFP vs. AAV-PERM1: 0.93 vs. 1.43 and 0.92 vs. 1.64 in DEF and DFS, respectively, both p<0.05 in t-test). In addition, we observed a subtle, yet significant increase in the ratio of the heart weight to tibial length in AAV-PERM1 mice (AAV-GFP vs. AAV-PERM1: 4.7 vs 5.97, p<0.05 in t-test), suggestive of moderate cardiac hypertrophy development. Furthermore, PERM1 overexpression in the heart increased the mitochondrial copy number by 38% as compared to control AAV-GFP mice (p<0.05 in t-test), concurrently with an upregulation of the mitochondrial bioenergetics master regulator PGC-1α (155% of control, p<0.05 in t-test). Overall, these results showed that AAV-mediated PERM1 overexpression simultaneously enhances muscle contractility and mitochondrial biogenesis in the heart. This study further suggests that the gene delivery of PERM1 could be a potential therapeutic approach to treat HFrEF patients.

We and others demonstrated that PERM1 is a positive regulator of mitochondrial bioenergetics in the heart. However, discrepant results have emerged with regard to whether PERM1 loss-of-function affect cardiac contractility. In order to exclude the possibility that the reported negative results can be due to insufficient power of statistical test, we conducted a more robust echocardiography (Echo) analysis by increasing the sample size. We used Perm1-KO and their respective wildtype (WT) littermates, which were destined to tissue harvest. This yielded 84 WT mice and 88 Perm1-KO mice. We analyzed Echo-derived parameters of left ventricular (LV) systolic function. At the end of the study, ejection fraction (EF) was 65.43 +/- 7.13 in WT vs. 53.98 +/- 8.80 in Perm1-KO yielding p < 0.00000000000000004. Other parameters which reached statistically significant difference between WT and Perm1-KO (at p < 0.05) included LV fractional shortening (FS), LV diastolic and systolic diameters, LV anterior and posterior systolic wall thickness, LV posterior wall systolic thickening, stroke volume, and cardiac output (CO). Retrospectively, a p value < 0.05 was consistently achieved in assessment of EF only after average N per group reached 13. Larger minimal N per group were required for other parameters. Of interest, in both groups there were no correlation between EF% and CO. At the same time, in both groups EF strongly inversely correlated with LV diastolic diameter. This led us to a speculation that low EF may be in part compensated by an increased LV circumference, for the purpose of maintaining invariant CO. Indeed, the intergroup difference in CO (6%) was much smaller than the intergroup difference in EF (18%). We conclude that PERM1 does regulate cardiac mechanics. Changes caused by constitutive Perm1-KO can be conceptualized as reduced contractility partially compensated by increased LV circumference. This study underscores the importance of sufficiently large sample size for detecting significant differences in Echo data. ### Competing Interest Statement The authors have declared no competing interest.

The tricarboxylic acid (TCA) cycle plays a crucial role in mitochondrial ATP production in the healthy heart. However, in heart failure, the TCA cycle becomes dysregulated. Understanding the mechanism by which TCA cycle genes are transcribed in the healthy heart is an important prerequisite to understanding how these genes become dysregulated in the failing heart. PGC-1α is a transcriptional coactivator that broadly induces genes involved in mitochondrial ATP production. PGC-1α potentiates its effects through coactivation of coupled transcription factors, such as ERR, Nrf1, Gabpa, and YY1. We hypothesized that PGC-1α plays an essential role in transcription of TCA cycle genes. Thus, by utilizing localization peaks of PGC-1α to TCA cycle gene promoters, it would allow the identification of coupled transcription factors. PGC-1α potentiated the transcription of 13 out of 14 TCA cycle genes, partly through ERR, Nrf1, Gabpa, and YY1. ChIP-sequencing showed PGC-1α localization peaks in TCA cycle gene promoters. Transcription factors with binding elements that were found proximal to PGC-1α peak localization were generally essential for transcription of the gene. These transcription factor binding elements were well conserved between mice and humans. Among the four transcription factors, ERR and Gabpa played a major role in potentiating transcription when compared to Nrf1 and YY1. These transcription factor-dependent PGC-1α recruitment was verified with Idh3a, Idh3g, and Sdha promoters with DNA binding assay. Taken together, this study clarifies the mechanism by which TCA cycle genes are transcribed, which could be useful to understand how those genes are dysregulated in pathological conditions.

O-GlcNAcylation (O-GlcNAc) is a post-translational modification of proteins where N-acetylglucosamines are added to serine and threonine residues in proteins. This modification is regulated by two enzymes: O-GlcNAc transferase (OGT) that adds O-GlcNAc to proteins; O-GlcNAcase (OGA) that removes O-GlcNAc from proteins. A recent study demonstrated that overexpression of OGT in the heart leads to excessive O-GlcNAcylation, resulting in mitochondrial dysfunction and the development of heart failure. However, what regulates OGT expression in the heart remains elusive. Perm1 is a striated-muscle-specific regulator of mitochondrial bioenergetics. Here, we found that Perm1 suppresses O-GlcNAcylation via gene repression of OGT. Adenovirus-mediated overexpression of Perm1 suppressed O-GlcNAcylation of proteins in H9c2 cells (47% of control, p<0.05), concurrent with downregulation of OGT (52% from control, p<0.05). Luciferase gene reporter assay revealed that Perm1 reduces the promoter activity of OGT, indicating that Perm1 acts as a gene repressor of OGT. Furthermore, phenylephrine-induced O-GlcNAcylation in cardiomyocytes was reversed by Perm1 overexpression through repressing OGT expression. Conversely, loss of Perm1 (Perm1-KO) in mice led to upregulation of OGT and the increase of O-GlcNAcylated proteins in the heart (176% and 144% from WT, respectively, both p<0.05, n=6 in WT, n=7 in KO). Concomitantly, Perm1-KO mice exhibited reduced contractility and mitochondrial function, manifested by a significant decrease in ejection fraction and succinate dehydrogenase activity, respectively (87% and 53% of WT, both p<0.05). Lastly, subcellular fractionation and co-immunoprecipitation assay of mouse hearts revealed that loss of Perm1 specifically increases O-GlcNAcylation of nuclear proteins, concurrent with excessive O-GlcNAcylation of PGC-1α, a master regulator of mitochondrial bioenergetics (2-fold increase in O-GlcNAcylated PGC-1α/total PGC-1α, p<0.05, n=3/group). Our results suggest that Perm1 is a novel regulator of O-GlcNAcylation in the heart that transcriptionally represses OGT expression, which presumably prevents excessive O-GlcNAcylation of PGC-1α and mitochondrial dysfunction under pathological stress.

O-GlcNAcylation is a post-translational modification of proteins that plays an important role in cellular homeostasis and stress responses. Two enzymes regulate O-GlcNAcylation: O-GlcNAc transferase (OGT) that adds O-GlcNAc to proteins; O-GlcNAcase (OGA) that removes O-GlcNAc from proteins. While O-GlcNAcylation is necessary to respond to ischemia/reperfusion injury, chronic activation of O-GlcNAcylation in the heart has adverse effects, and excessive O-GlcNAcylation leads to the development of heart failure. However, there is currently no therapy for heart failure that targets O-GlcNAcylation. Perm1 is a striated muscle-specific regulator of mitochondrial bioenergetics. We previously demonstrated that Perm1-knockout mice exhibit reduced cardiac function and myocardial energy reserve, in association with excessive O-GlcNAcylation and upregulation of OGT. Here, we hypothesized that Perm1 maintains mitochondrial energetics by suppressing O-GlcNAcylation. We found that adenovirus-mediated overexpression of Perm1 in cardiomyocytes significantly decreased O-GlcNAcylation (Figure 1A-B) and increased the basal and maximal respiration and ATP production rates as compared with control in Cell Mito Stress Test using a Seahorse 96x flux analyzer (orange vs. green, Figure 1C-D). Furthermore, the increased levels of O-GlcNAcylated proteins (215% of control, p<0.05) via silencing OGA (si-OGA) in cardiomyocytes significantly decreased the basal and maximal respiration capacity and ATP production rates as compared with control (green vs. purple, Figure 1C-D), all which were completely rescued by Perm1 overexpression (blue, Figure 1C-D). These results suggest that Perm1 positively regulates mitochondrial energetics, in part, via suppressing O-GlcNAcylation and that Perm1 might be a new therapeutic target of heart failure that maintains mitochondrial function through preventing excessive O-GlcNAcylation under pathological stress.

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.

BACKGROUND: PRDM16 plays a role in myocardial development through TGF-β (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 Prdm16 Q187X 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-β–associated transcripts. Homozygous Prdm16 Q187X/Q187X mice demonstrated an underdeveloped compact myocardium and were embryonically lethal. Heterozygous Prdm16 Q187X/WT mice demonstrated significantly smaller ventricular dimensions, heightened fibrosis, and age-dependent loss of TGF-β 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-β signaling.

The heart is a metabolically demanding organ that requires significant amounts of energy to function properly, and even minor metabolic deficiencies during development can lead to heart failure. Cardiomyopathies are a group of conditions that affect the heart's contractile function, and PRDM16 deficiency is a primary cause of cardiomyopathies associated with 1p36 deletion syndrome in humans. Conditional deletion of Prmd16 in the heart causes a spectrum of cardiac phenotypes including left ventricular non-compaction, late-onset heart failure, and dilated cardiomyopathy. Still, the mechanisms underlying these cardiac phenotypes with loss of Prdm16 are not fully understood. We and others demonstrated that lack of Prdm16 in the heart causes alterations in fatty acid (FA) oxidation and oxidative phosphorylation (OXPHOS) gene expression. Further, we now show that Prdm16 cardiac deletion reduces the expression of key regulators of FA oxidation and OXPHOS genes including Perm1. Loss and gain of Prdm16 function in vitro confirmed a cell-autonomous regulation of Perm1 by Prdm16 in H9c2 rat cardiomyoblasts and neonatal rat ventricular myocytes (NRVMs). Prdm16 ChIP-sequencing analysis of embryonic hearts revealed direct binding of Prdm16 to the promoter of Perm1. Prdm16 binding to Perm1 promoter was further confirmed by ChIP-qPCR in H9c2 cells. Knockdown of Prdm16 in NRVMs reduced maximal mitochondrial respiration, which was rescued by over-expressing Perm1. Further, PERM1 mRNA was diminished in human induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) derived from a patient with a PRDM16 mutation (Q187X). These results are evidence that PRDM16 is an upstream regulator of PERM1 in the heart.

Some missense gain-of-function mutations in the CACNA1C gene, encoding calcium channel CaV1.2, cause a life-threatening form of long QT syndrome (LQTS) 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 induced pluripotent stem cell (iPSC)-derived cardiomyocytes generated from patients with Timothy syndrome and two common forms of LQTS, type 1 (LQTS1) and type 2 (LQTS2), caused by missense trafficking mutations in potassium channels. Electrophysiological recordings demonstrate that a Food and Drug Administration (FDA)-approved cough suppressant, dextromethorphan, can be used as an agonist of SIGMAR1 to shorten the prolonged action potential in cardiomyocytes from patients with Timothy syndrome and human cellular models of LQTS1 and LQTS2. When tested in vivo, dextromethorphan also normalized the prolonged QT intervals in a mouse model of Timothy syndrome. Overall, our study demonstrates that SIGMAR1 is a potential therapeutic target for Timothy syndrome and possibly other inherited arrhythmias such as LQTS1 and LQTS2. Song and colleagues show that FDA-approved cough suppressant dextromethorphan could be used as an agonist of sigma non-opioid intracellular receptor 1 (SIGMAR1) to normalize the action potential in human cellular models and a mouse model of Timothy syndrome, a congenital disease with no available treatment. The researchers also show that dextromethorphan normalizes the action potential in human cellular models of two additional inherited cardiac arrhythmias: long QT syndrome types 1 and 2, which are caused by mutations in different genes.

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.

Background. Perm1 is a striated-muscle specific protein that is predominantly expressed in cardiac and skeletal muscle. We recently demonstrated that Perm1 is downregulated in the human and mouse failing hearts. However, whether and how Perm1 regulates cardiac function remains unknown. Beta-O-linkage of N-acetylglucosamine (O-GlcNAc) post-translational modification plays a role in stress response, and the excessive O-GlcNAc in the heart leads to the development of heart failure. We hypothesize that Perm1 regulates cardiac function through O-GlcNAcylation. Methods and Results. Our newly generated Perm1-knockout (KO) mice exhibited reduced cardiac function as compared to their wild-type (WT) littermates (50.1% vs. 34.7% in ejection fraction, p<0.05, n=15/group), which was associated with a significant increase in the total levels of O-GlcNAcylated proteins (139% from WT, p<0.05, n=6 in WT, n=8 in KO). To determine whether Perm1 downregulation increases the vulnerability to pressure overload, WT and Perm1 heterozygous (Perm1 +/- ) mice were subjected to transverse aortic constriction (TAC) surgery for 1 week. The total O-GlcNAcylated protein levels were markedly increased in Perm1 +/- -TAC mice as compared to WT-TAC mice (549% vs. 173% from non-TAC WT, p<0.05, Fig.1A, B), concurrent with lower ejection fraction than WT-TAC mice (50.0% vs. 41.5%, p=0.06, n=5 in WT-TAC, n=3 in Perm1 +/- -TAC). The excessive O-GlcNAc in Perm1 +/- mice was associated with upregulation of O-GlcNAc transferase (OGT, the enzymes that adds O-GlcNAc to proteins, p<0.05, Fig1A, C), while there was no significant difference in the expression of O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc from proteins) in WT vs. Perm1 +/- mice in post-TAC (Fig.1A, D). Conclusions. These data suggest that Perm1 suppresses excessive O-GlcNAcylation occurring in a stressed heart through regulating OGT expression. Hence, the maintenance of Perm1 expression may be protective in the failing heart.

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.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.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.

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 ERR alpha and its coactivator PGC-1 alpha 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 ERR alpha, we performed co-immunoprecipitation assays, which showed that PERM1 bound to ERR alpha 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 ERR alpha. 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-1 alpha, 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 ERR alpha/PGC-1 alpha axis that functionally interacts with BAG6 and KANK2.

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.

Rationale: Diabetic cardiomyopathy is accompanied by increased production of NADH, predominantly through oxidation of fatty acids and consequent increases in oxidative stress. The role of Nampt (nicotinamide phosphoribosyltransferase), the rate-limiting enzyme of the salvage pathway of nicotinamide adenine dinucleotide synthesis, in the development of diabetic cardiomyopathy is poorly understood. Objective: We investigated the role of endogenous and exogenous Nampt during the development of diabetic cardiomyopathy in response to high-fat diet (HFD) consumption and in the context of oxidative stress. Methods and Results: HFD consumption upregulated endogenous Nampt, and HFD-induced cardiac diastolic dysfunction, fibrosis, apoptosis, and proinflammatory signaling were alleviated in transgenic mice with cardiac-specific overexpression of Nampt. The alleviation of diastolic dysfunction observed in these mice was abolished by inhibition of NADP(H) production via NADK (NAD kinase) inhibition. Nampt overexpression decreased oxidation of GSH (glutathione) and Trx1 (thioredoxin 1) targets, dityrosine, and the accumulation of toxic lipids, including ceramides and diglycerides, in the presence of HFD consumption. Nampt overexpression upregulated not only NAD + (nicotinamide adenine dinucleotide) but also NADP + (nicotinamide adenine dinucleotide phosphate) and NADPH in the heart and in cultured cardiomyocytes, which, in turn, stimulated the glutathione and Trx1 systems and alleviated oxidative stress in the heart induced by HFD consumption. In cultured cardiomyocytes, Nampt-induced upregulation of NADPH was abolished in the presence of NADK knockdown, whereas that of NAD + was not. Nampt overexpression attenuated H 2 O 2 -induced oxidative inhibition of Prdx1 (peroxiredoxin 1) and mTOR (mammalian target of rapamycin) in an NADK-dependent manner in cultured cardiomyocytes. Nampt overexpression also attenuated H 2 O 2 -induced cell death, an effect that was partly abolished by inhibition of NADK, Trx1, or glutathione synthesis. In contrast, oxidative stress and the development of diabetic cardiomyopathy in response to HFD consumption were exacerbated in Nampt heterozygous knockout (Nampt +/− ) mice. Conclusions: Nampt-mediated production of NAD + protects against oxidative stress, in part, through the NADPH-dependent reducing system, thereby alleviating the development of diabetic cardiomyopathy in response to HFD consumption.