Cardiac-specific Knockout Mice Research Articles

PKP2 gene therapy reduces ventricular arrhythmias, reverses ventricular remodeling, improves heart function, and reduces mortality in a mouse model of arrhythmogenic right ventricular cardiomyopathy

Abstract Introduction Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Currently, there are no approved treatments that address the underlying genetic cause of this disease, representing a significant unmet need. Purpose Mutations in desmosome gene Plakophilin-2 (PKP2) account for approximately 40% of ARVC cases and result in reduced gene expression. Our goal is to examine the feasibility and the efficacy of restoration of PKP2 expression in a cardiac specific knock-out mouse model of Pkp2. Methods Adeno-associated virus 9 (AAV9) was used to deliver a wild-type copy of PKP2 gene that is driven by a cardiac-selective promoter and expressed selectively in cardiomyocytes. Results We demonstrated that a single-dose AAV9-mediated PKP2 gene therapy effectively prevented disease onset before overt cardiomyopathy and attenuated disease progression after overt cardiomyopathy. Restoration of PKP2 expression led to a significant extension of lifespan by restoring cellular structures of desmosomes and gap junctions, preventing or halting decline in left ventricular ejection fraction, preventing or reversing dilation of the right ventricle, attenuating ventricular arrhythmia event frequency and severity, and preventing adverse fibrotic remodeling. Additional RNA sequencing analyses showed that durable restoration of PKP2 expression led to highly coordinated and durable correction of PKP2-associated transcriptional networks beyond desmosomes, revealing a broad spectrum of biological perturbances behind ARVC disease etiology. Conclusion These results indicate that a cardiac-selective AAV9-mediated PKP2 gene therapy may be a promising one-time treatment for ARVC patients with PKP2 mutations.

Sodium-glucose exchanger 2 inhibitor canagliflozin promotes mitochondrial metabolism and alleviates salt-induced cardiac hypertrophy via preserving SIRT3 expression

IntroductionExcess salt intake is not only an independent risk factor for heart failure, but also one of the most important dietary factors associated with cardiovascular disease worldwide. Metabolic reprogramming in cardiomyocytes is an early event provoking cardiac hypertrophy that leads to subsequent cardiovascular events upon high salt loading. Although SGLT2 inhibitors, such as canagliflozin, displayed impressive cardiovascular health benefits, whether SGLT2 inhibitors protect against cardiac hypertrophy-related metabolic reprogramming upon salt loading remain elusive. ObjectivesTo investigate whether canagliflozin can improve salt-induced cardiac hypertrophy and the underlying mechanisms. MethodsDahl salt-sensitive rats developed cardiac hypertrophy by feeding them an 8% high-salt diet, and some rats were treated with canagliflozin. Cardiac function and structure as well as mitochondrial function were examined. Cardiac proteomics, targeted metabolomics and SIRT3 cardiac-specific knockout mice were used to uncover the underlying mechanisms. ResultsIn Dahl salt-sensitive rats, canagliflozin showed a potent therapeutic effect on salt-induced cardiac hypertrophy, accompanied by lowered glucose uptake, reduced accumulation of glycolytic end-products and improved cardiac mitochondrial function, which was associated with the recovery of cardiac expression of SIRT3, a key mitochondrial metabolic regulator. Cardiac-specific knockout of SIRT3 not only exacerbated salt-induced cardiac hypertrophy but also abolished the therapeutic effect of canagliflozin. Mechanistically, high salt intake repressed cardiac SIRT3 expression through a calcium-dependent epigenetic modifications, which could be blocked by canagliflozin by inhibiting SGLT1-mediated calcium uptake. SIRT3 improved myocardial metabolic reprogramming by deacetylating MPC1 in cardiomyocytes exposed to pro-hypertrophic stimuli. Similar to canagliflozin, the SIRT3 activator honokiol also exerted therapeutic effects on cardiac hypertrophy. ConclusionCardiac mitochondrial dysfunction caused by SIRT3 repression is a critical promotional determinant of metabolic pattern switching underlying salt-induced cardiac hypertrophy. Improving SIRT3-mediated mitochondrial function by SGLT2 inhibitors-mediated calcium handling would represent a therapeutic strategy against salt-related cardiovascular events.

AAV9:PKP2 improves heart function and survival in a Pkp2-deficient mouse model of arrhythmogenic right ventricular cardiomyopathy

BackgroundArrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Currently, there are no approved treatments that address the underlying genetic cause of this disease, representing a significant unmet need. Mutations in Plakophilin-2 (PKP2), encoding a desmosomal protein, account for approximately 40% of ARVC cases and result in reduced gene expression.MethodsOur goal is to examine the feasibility and the efficacy of adeno-associated virus 9 (AAV9)-mediated restoration of PKP2 expression in a cardiac specific knock-out mouse model of Pkp2.ResultsWe show that a single dose of AAV9:PKP2 gene delivery prevents disease development before the onset of cardiomyopathy and attenuates disease progression after overt cardiomyopathy. Restoration of PKP2 expression leads to a significant extension of lifespan by restoring cellular structures of desmosomes and gap junctions, preventing or halting decline in left ventricular ejection fraction, preventing or reversing dilation of the right ventricle, ameliorating ventricular arrhythmia event frequency and severity, and preventing adverse fibrotic remodeling. RNA sequencing analyses show that restoration of PKP2 expression leads to highly coordinated and durable correction of PKP2-associated transcriptional networks beyond desmosomes, revealing a broad spectrum of biological perturbances behind ARVC disease etiology.ConclusionsWe identify fundamental mechanisms of PKP2-associated ARVC beyond disruption of desmosome function. The observed PKP2 dose-function relationship indicates that cardiac-selective AAV9:PKP2 gene therapy may be a promising therapeutic approach to treat ARVC patients with PKP2 mutations.

Abstract 13568: Proteomic Analysis Reveals Protein Alterations in Drp1 Cardiac Knockout Mice With Circadian Rhythm Disruption

Introduction: Mitochondria dynamics are altered in circadian rhythm disruption, and dynamin-related protein 1 (Drp1) may be an important target. Methods: Wild-type and Drp1 cardiac-specific knockout mice were divided into normal circadian group and circadian disorder group, respectively. Normal circadian group mice were housed in a standard environment (12 hours of light/12 hours of darkness), while dark group mice were housed in a 24-hour dark environment. After 4 weeks, the cardiac tissues were analysed using Tandem Mass Tag proteomics. Differentially expressed proteins (DEPs) was identified. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. Gene set enrichment analysis (GSEA) was performed to find enriched biological processes. Results: Compared with the WT/N group, the Drp1-/N group possessed a total of 176 DEPs. These proteins are widely involved in mitochondrial membrane and metabolism process. We identified several differentially expressed mitochondrial dynamics-related genes, including Drp1, using Western blotting. Compared with the WT/D group, the Drp1-/D group exhibited a total of 126 DEPs. GO and KEGG showed that the DEPs were mainly involved in response to stress, metabolic pathways. For Drp1 cKO mice, the protein changes caused by the circadian rhythm disorder were mainly concentrated in vitamin transport, complement coagulation cascades. Conclusions: This study may provide an insight into the myocardial protein changes caused by Drp1 cKO with circadian rhythm disruption, which may involve in terms such as metabolic pathways, complement coagulation, and vitamin transport.

RBPMS regulates cardiomyocyte contraction and cardiac function through RNA alternative splicing.

RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.

The CREG1-FBXO27-LAMP2 axis alleviates diabetic cardiomyopathy by promoting autophagy in cardiomyocytes

Autophagy plays an important role in the development of diabetic cardiomyopathy. Cellular repressor of E1A-stimulated genes 1 (CREG1) is an important myocardial protective factor. The aim of this study was to investigate the effects and mechanisms of CREG1 in diabetic cardiomyopathy. Male C57BL/6 J mice, Creg1 transgenic mice and cardiac-specific knockout mice were used to establish a type 2 diabetes model. Small animal ultrasound, Masson’s staining and western blotting were used to evaluate cardiac function, myocardial fibrosis and autophagy. Neonatal mouse cardiomyocytes (NMCMs) were stimulated with palmitate, and the effects of CREG1 on NMCMs autophagy were examined. CREG1 deficiency exacerbated cardiac dysfunction, cardiac hypertrophy and fibrosis in mice with diabetic cardiomyopathy, which was accompanied by exacerbated autophagy dysfunction. CREG1 overexpression improved cardiac function and ameliorated cardiac hypertrophy and fibrosis in diabetic cardiomyopathy by improving autophagy. CREG1 protein expression was decreased in palmitate-induced NMCMs. CREG1 knockdown exacerbated cardiomyocyte hypertrophy and inhibited autophagy. CREG1 overexpression inhibited cardiomyocyte hypertrophy and improved autophagy. LAMP2 overexpression reversed the effect of CREG1 knockdown on palmitate-induced inhibition of cardiomyocyte autophagy. CREG1 inhibited LAMP2 protein degradation by inhibiting the protein expression of F-box protein 27 (FBXO27). Our findings indicate new roles of CREG1 in the development of diabetic cardiomyopathy.

Mitochondrial GSNOR Alleviates Cardiac Dysfunction via ANT1 Denitrosylation.

The cardiac-protective role of GSNOR (S-nitrosoglutathione reductase) in the cytoplasm, as a denitrosylase enzyme of S-nitrosylation, has been reported in cardiac remodeling, but whether GSNOR is localized in other organelles and exerts novel effects remains unknown. We aimed to elucidate the effects of mitochondrial GSNOR, a novel subcellular localization of GSNOR, on cardiac remodeling and heart failure (HF). GSNOR subcellular localization was observed by cellular fractionation assay, immunofluorescent staining, and colloidal gold particle staining. Overexpression of GSNOR in mitochondria was achieved by mitochondria-targeting sequence-directed adeno-associated virus 9. Cardiac-specific knockout of GSNOR mice was used to examine the role of GSNOR in HF. S-nitrosylation sites of ANT1 (adenine nucleotide translocase 1) were identified using biotin-switch and liquid chromatography-tandem mass spectrometry. GSNOR expression was suppressed in cardiac tissues of patients with HF. Consistently, cardiac-specific knockout mice showed aggravated pathological remodeling induced by transverse aortic constriction. We found that GSNOR is also localized in mitochondria. In the angiotensin II-induced hypertrophic cardiomyocytes, mitochondrial GSNOR levels significantly decreased along with mitochondrial functional impairment. Restoration of mitochondrial GSNOR levels in cardiac-specific knockout mice significantly improved mitochondrial function and cardiac performance in transverse aortic constriction-induced HF mice. Mechanistically, we identified ANT1 as a direct target of GSNOR. A decrease in mitochondrial GSNOR under HF leads to an elevation of S-nitrosylation ANT1 at cysteine 160 (C160). In accordance with these findings, overexpression of either mitochondrial GSNOR or ANT1 C160A, non-nitrosylated mutant, significantly improved mitochondrial function, maintained the mitochondrial membrane potential, and upregulated mitophagy. We identified a novel species of GSNOR localized in mitochondria and found mitochondrial GSNOR plays an essential role in maintaining mitochondrial homeostasis through ANT1 denitrosylation, which provides a potential novel therapeutic target for HF.

Lysine crotonylation of SERCA2a correlates to cardiac dysfunction and arrhythmia in Sirt1 cardiac-specific knockout mice

Protein post-translational modifications (PTMs) are important regulators of protein functions and produce proteome complexity. SIRT1 has NAD+-dependent deacylation of acyl-lysine residues. The present study aimed to explore the correlation between lysine crotonylation (Kcr) on cardiac function and rhythm in Sirt1 cardiac-specific knockout (ScKO) mice and related mechanism. Quantitative proteomics and bioinformatics analysis of Kcr were performed in the heart tissue of ScKO mice established with a tamoxifen-inducible Cre-loxP system. The expression and enzyme activity of crotonylated protein were assessed by western blot, co-immunoprecipitation, and cell biology experiment. Echocardiography and electrophysiology were performed to investigate the influence of decrotonylation on cardiac function and rhythm in ScKO mice. The Kcr of SERCA2a was significantly increased on Lys120 (1.973 folds). The activity of SERCA2a decreased due to lower binding energy of crotonylated SERCA2a and ATP. Changes in expression of PPAR-related proteins suggest abnormal energy metabolism in the heart. ScKO mice had cardiac hypertrophy, impaired cardiac function, and abnormal ultrastructure and electrophysiological activities. We conclude that knockout of SIRT1 alters the ultrastructure of cardiac myocytes, induces cardiac hypertrophy and dysfunction, causes arrhythmia, and changes energy metabolism by regulating Kcr of SERCA2a. These findings provide new insight into the role of PTMs in heart diseases.

NUCLEOLIN PROMOTES AUTOPHAGY THROUGH PGC-1Α IN LPS-INDUCED MYOCARDIAL INJURY.

As a multifunctional protein, nucleolin can participate in a variety of cellular processes. Nucleolin also has multiple protective effects on heart disease. Previous studies have shown that nucleolin could not only resist oxidative stress damage and inflammatory damage, but also regulate autophagy to play a protective role in cardiac ischemia. However, the specific mechanism has not been fully elucidated in LPS-induced myocardial injury. Therefore, the aim of this study is to explore the underlying mechanism by which nucleolin regulates autophagy to protect against LPS-induced myocardial injury in vivo and in vitro . In our study, we found that nucleolin could bind to PGC-1α, and we predicted that this interaction could promote autophagy and played a role in inhibiting cardiomyocyte apoptosis. Downregulation of nucleolin in H9C2 cells resulted in decreased autophagy and increased cell apoptosis during LPS-induced myocardial injury, while upregulation of PGC-1α had the opposite protective effect. Upregulation of nucleolin expression in cardiomyocytes could increase the level of autophagy during LPS-induced myocardial injury. In contrast, interference with PGC-1α expression resulted in a decrease in the protective effect of nucleolin, leading to reduced autophagy and thus increasing apoptosis. By using tandem fluorescent-tagged LC3 autophagic flux detection system, we observed autophagic flux and determined that PGC-1α interference could block autophagic lysosomal progression. We further tested our hypothesis in the nucleolin cardiac-specific knockout mice. Finally, we also found that inhibition of autophagy can reduce mitochondrial biogenesis as well as increase apoptosis, which demonstrated the importance of autophagy. Therefore, we can speculate that nucleolin can protect LPS-induced myocardial injury by regulating autophagy, and this protective effect may be mediated by the interaction with PGC-1α, which can positively regulate the ULK1, an autophagy-related protein. Our study provides a new clue for the cardioprotective effect of nucleolin, and may provide new evidence for the treatment of LPS-induced myocardial injury through the regulation of autophagy.

FAM122A Is Required for Mesendodermal and Cardiac Differentiation of Embryonic Stem Cells.

Mesendodermal specification and cardiac differentiation are key issues for developmental biology and heart regeneration medicine. Previously, we demonstrated that FAM122A, a highly conserved housekeeping gene, is an endogenous inhibitor of protein phosphatase 2A (PP2A) and participates in multifaceted physiological and pathological processes. However, the in vivo function of FAM122A is largely unknown. In this study, we observed that Fam122 deletion resulted in embryonic lethality with severe defects of cardiovascular developments and significantly attenuated cardiac functions in conditional cardiac-specific knockout mice. More importantly, Fam122a deficiency impaired mesendodermal specification and cardiac differentiation from mouse embryonic stem cells but showed no influence on pluripotent identity. Mechanical investigation revealed that the impaired differentiation potential was caused by the dysregulation of histone modification and Wnt and Hippo signaling pathways through modulation of PP2A activity. These findings suggest that FAM122A is a novel and critical regulator in mesendodermal specification and cardiac differentiation. This research not only significantly extends our understanding of the regulatory network of mesendodermal/cardiac differentiation but also proposes the potential significance of FAM122A in cardiac regeneration.

Abstract 10586: CREG1-FBXO27-LAMP2 Axis Promotes Autophagy Function of Cardiomyocytes and Alleviates Diabetic Cardiomyopathy

Introduction: Diabetic cardiomyopathy is a chronic complication of diabetes in the absence of coronary artery disease, arterial hypertension and valvular disease. Autophagy plays an important role in the development of diabetic cardiomyopathy. Cellular repressor of E1A-stimulated genes 1 (CREG1) is an important myocardial protective factor. The aim of this study was to investigate the effects and mechanisms of CREG1 in diabetic cardiomyopathy. Methods: Male C57BL/6J mice, CREG1 transgenic mice, and CREG1 cardiac-specific knockout mice were used to establish a type 2 diabetic model. Small animal ultrasound, Masson’s staining, and western blotting were used to evaluate cardiac function, myocardial fibrosis and autophagy. Neonatal mouse cardiomyocytes (NMCMs) were isolated and cultured. NMCMs were infected with CREG1- overexpressing adenovirus or small interfering RNA, followed by stimulation with palmitic acid (PA), real-time PCR, western blotting and immunofluorescence staining were used. Results: CREG1 protein expression was decreased in the myocardium of diabetic cardiomyopathy. CREG1 deficiency in the heart aggravated cardiac dysfunction, cardiac hypertrophy, and fibrosis in mice with diabetic cardiomyopathy, accompanied by aggravated autophagy dysfunction. CREG1 overexpression improved cardiac function and ameliorated cardiac hypertrophy and fibrosis in diabetic cardiomyopathy, by improving autophagy. CREG1 protein expression was decreased in PA-stimulated NMCMs. CREG1 knockdown aggravated cardiomyocyte hypertrophy and inhibited autophagy, which were reversed by resveratrol treatment. Conversely, CREG1 overexpression inhibited cardiomyocyte hypertrophy and improved autophagy, which were reversed by chloroquine or bafilomycin A1 treatment. Overexpression of LAMP2 reversed the effect of CREG1 knockdown on the PA-induced inhibition of cardiomyocyte autophagy. CREG1 inhibited the LAMP2 protein degradation by inhibiting the expression of F-box protein 27 (FBXO27). Conclusions: CREG1-FBXO27-LAMP2 axis alleviated diabetic cardiomyopathy by promoting cardiomyocyte autophagy. Our findings might help clarify new roles of CREG1 in the development of diabetic cardiomyopathy.

Abstract 12581: The N 6 -Methyladenosine Methylase Wtap is Essential for Cardiac Homeostasis

Introduction: N 6 -methyladenosine (m 6 A) methylation is one of the most common internal modifications in mammalian mRNA, and WTAP is a critical regulatory subunit of the RNA N 6 -methyladenosine methyltransferase complex. However, the roles of WTAP in cardiac structure and function are rarely known. Methods and Results: We found that the levels of m 6 A RNA methylation and the expression of WTAP were both decreased in myocardial tissue of the patients with hypertrophic cardiomyopathy, as well as in pressure overload-induced cardiac hypertrophy in mice. To determine the regulatory roles of WTAP, a conditional cardiac-specific knockout mice of WTAP was generated through Tamoxifen-inducible αMHC-Cre/loxP system. Depletion of WTPA resulted in heart failure in five days after Tamoxifen treatment, shown by enlarged heart and decreased left ventricular ejection fraction. Furthermore, we found WTAP knockout aggravated the progress of pathogenic cardiac remodeling induced by pressure overload, and abrogated the compensated response cardiac hypertrophy to hypertrophic stimulation. We found MYLK4 was a direct target of WTAP through a methylated RNA immunoprecipitation (MeRIP) sequencing, and it was validated by MeRIP-qPCR, RT-qPCR and RIP-qPCR. RNA stability analysis and dual-luciferase assays showed that depression of WTAP decreased m6A level of MYLK4 mRNA and resulted in a down-regulation of MYLK4 by reducing its RNA-stability. Forced overexpression of MYLK4 adeno-associated virus was able to partly alleviate the cardiac dysfunction induced by WTAP deletion. Finally, it was surprising that overexpression of WTAP also promotes pathogenic cardiomyocyte hypertrophy both in vitro and in vivo, as well as exacerbated cardiac remolding induced by pressure overload. Conclusions: In conclusion, our study demonstrates that WTAP is a critical regulator of cardiac function and indicates that appropriate m 6 A RNA methylation is required for cardiac homeostasis.

The E3 Ligase TRIM16 Is a Key Suppressor of Pathological Cardiac Hypertrophy.

Pathological cardiac hypertrophy is one of the leading causes of heart failure with highly complicated pathogeneses. The E3 ligase TRIM16 (tripartite motif-containing protein 16) has been recognized as a pivotal regulator to control cell survival, immune response, and oxidativestress. However, the role of Trim16 in cardiac hypertrophy is unknown. We generated cardiac-specific knockout mice and adeno-associated virus serotype 9-Trim16 mice to evaluate the function of Trim16 in pathological myocardial hypertrophy. The direct effect of TRIM16 on cardiomyocyte enlargement was examined using an adenovirus system. Furthermore, we combined RNA-sequencing and interactome analysis that was followed by multiple molecular biological methodologies to identify the direct target and corresponding molecular events contributing to TRIM16 function. We found an intimate correlation of Trim16 expression with hypertrophy-related heart failure in both human and mouse. Our functional investigations and unbiased transcriptomic analyses clearly demonstrated that Trim16 deficiency markedly exacerbated cardiomyocyte enlargement in vitro and in transverse aortic constriction-induced cardiac hypertrophy mouse model, whereas Trim16 overexpression attenuated cardiac hypertrophy and remodeling. Mechanistically, Prdx1 (peroxiredoxin 1) is an essential target of Trim16 in cardiac hypertrophy. We found that Trim16 interacts with Prdx1 and inhibits its phosphorylation, leading to a robust enhancement of its downstream Nrf2 (nuclear factor-erythroid 2-related factor 2) pathway to block cardiac hypertrophy. Trim16-blocked Prdx1 phosphorylation was largely dependent on a direct interaction between Trim16 and Src and the resultant Src ubiquitinational degradation. Notably, Prdx1 knockdown largely abolished the anti-hypertrophic effects of Trim16 overexpression. Our findings provide the first evidence supporting Trim16 as a novel suppressor of pathological cardiac hypertrophy and indicate that targeting the Trim16-Prdx1 axis represents a promising therapeutic strategy for hypertrophy-related heart failure.

Cereblon contributes to cardiac dysfunction by degrading Cav1.2α.

Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that was reported to target ion channel proteins. L-type voltage-dependent Ca2+ channel (LTCC) density and dysfunction is a critical player in heart failure with reduced ejection fraction (HFrEF). However, the underlying cellular mechanisms by which CRBN regulates LTCC subtype Cav1.2α during cardiac dysfunction remain unclear. Here, we explored the role of CRBN in HFrEF by investigating the direct regulatory role of CRBN in Cav1.2α activity and examining how it can serve as a target to address myocardial dysfunction. Cardiac tissues from HFrEF patients exhibited increased levels of CRBN compared with controls. In vivo and ex vivo studies demonstrated that whole-body CRBN knockout (CRBN-/-) and cardiac-specific knockout mice (Crbnfl/fl/Myh6Cre+) exhibited enhanced cardiac contractility with increased LTCC current (ICaL) compared with their respective controls, which was modulated by the direct interaction of CRBN with Cav1.2α. Mechanistically, the Lon domain of CRBN directly interacted with the N-terminal of Cav1.2α. Increasing CRBN levels enhanced the ubiquitination and proteasomal degradation of Cav1.2α and decreased ICaL. In contrast, genetic or pharmacological depletion of CRBN via TD-165, a novel PROTAC-based CRBN degrader, increased surface expression of Cav1.2α and enhanced ICaL. Low CRBN levels protected the heart against cardiomyopathy in vivo. Cereblon selectively degrades Cav1.2α, which in turn facilitates cardiac dysfunction. A targeted approach or an efficient method of reducing CRBN levels could serve as a promising strategy for HFrEF therapeutics.

Cardiac-specific LRP6 knockout induces lipid accumulation through Drp1/CPT1b pathway in adult mice.

We recently reported low-density lipoprotein receptor-related protein 6 (LRP6) decreased in dilated cardiomyopathy hearts, and cardiac-specific knockout mice displayed lethal heart failure through activation of dynamin-related protein 1 (Drp1). We also observed lipid accumulation in LRP6 deficiency hearts, but the detailed molecular mechanisms are unclear. Here, we detected fatty acids components in LRP6 deficiency hearts and explored the potential molecular mechanisms. Fatty acid analysis by GC-FID/MS revealed cardiac-specific LRP6 knockout induced the higher level of total fatty acids and some medium-long-chain fatty acids (C16:0, C18:1n9 and C18:2n6) than in control hearts. Carnitine palmitoyltransferase 1b (CPT1b), a rate-limiting enzyme of mitochondrial β-oxidation in adult heart, was sharply decreased in LRP6 deficiency hearts, coincident with the activation of Drp1. Drp1 inhibitor greatly improved cardiac dysfunction and attenuated the increase in total fatty acids and fatty acids C16:0, C18:1n9 in LRP6 deficiency hearts. It also greatly inhibited the decrease in the cardiac expression of CPT1b and the transcriptional factors CCCTC-binding factor (CTCF) and c-Myc induced by cardiac-specific LRP6 knockout in mice. C-Myc but not CTCF was identified to regulate CPT1b expression and lipid accumulation in cardiomyocytes in vitro. The present study indicated cardiac-specific LRP6 knockout induced lipid accumulation by Drp1/CPT1b pathway in adult mice, and c-Myc is involved in the process. It suggests that LRP6 regulates fatty acid metabolism in adult heart.

Abstract 274: Mitochondrial Translation Machinery Defects Causes Cardiomyopathy and i n vivo Functional Screening of Therapeutic Targets

More than 95% of ATP consumed in the heart is derived from oxidative phosphorylation (OxPhos) in the mitochondria. Besides over a thousand proteins encoded by nuclear DNA, mitochondrial gene expression machinery generates 13 proteins for the OxPhos system that employs mitochondrial ribosomal proteins for translation. Mitochondrial ribosome protein S5 (MRPS5) encoded by MRPs gene family, is located at the mammalian mitochondrial ribosome 28S subunit and its importance has not been explored in the heart. Western blotting showed that MRPS5 expression was decreased in TAC-induced hypertrophic mouse hearts ( in vivo ), and in phenylephrine or isoproterenol-stimulated cardiomyocytes ( in vitro ). In the failing hearts of patients, Mrps5-mediated mt-CO1 was markedly decreased as well. To determine the functional role of MRPS5 in the heart, we generated an inducible cardiac-specific knockout mouse and showed that Mrps5-deficient mice developed time-dependent cardiac hypertrophy, fibrosis and heart failure. Mitochondria in the Mrps5-deficient adult cardiomyocytes exhibited membrane swelling and cristae collapsed together with decreased oxygen consumption and ATP production. Mitochondrial calcium homeostasis was also disrupted. Combined analysis of transcriptomics and metabolomics spatiotemporally identified 20 potential target genes associated with Mrps5 deficiency-mediated cardiac pathological processes. Through in vivo functional screening via adeno-associated virus (AAV) mediated gene delivery, we further narrowed down and confirmed that Kruppel-like factor 15 (Klf15) remarkably rescued cardiac phenotype in the Mrps5-deficient mice with maintained mitochondrial homeostasis, suppressed cardiac hypertrophy and fibrosis, as well as preserved cardiac function. Further RNA-sequencing and biochemical analysis mechanistically revealed that genes involved in glycolysis/ gluconeogenesis, PPAR signaling pathway and biosynthesis of amino acids, markedly altered when exploited AAV-Klf15 gene therapy in Mrps5-deficient mice heart. Our results demonstrates Mrps5 is essential for mitochondrial homeostasis and cardiac function and uncovers Klf15 as a potential therapeutic target for treating cardiac mitochondrial diseases.

Ufm1-Specific Ligase Ufl1 Regulates Endoplasmic Reticulum Homeostasis and Protects Against Heart Failure.

Defects in protein homeostasis are sufficient to provoke cardiac remodeling and dysfunction. Although posttranslational modifications by ubiquitin and ubiquitin-like proteins are emerging as an important regulatory mechanism of protein function, the role of Ufm1 (ubiquitin-fold modifier 1)-a novel ubiquitin-like protein-has not been explored in either the normal or stressed heart. Western blotting revealed that Ufl1 (Ufm1-specific E3 ligase 1)-an enzyme essential for Ufm1 modification-was increased in hypertrophic mouse hearts but reduced in the failing hearts of patients with dilated cardiomyopathy. To determine the functional role of Ufl1 in the heart, we generated a cardiac-specific knockout mouse and showed that Ufl1-deficient mice developed age-dependent cardiomyopathy and heart failure, as indicated by elevated cardiac fetal gene expression, increased fibrosis, and impaired cardiac contractility. When challenged with pressure overload, Ufl1-deficient hearts exhibited remarkably greater hypertrophy, exacerbated fibrosis, and worsened cardiac contractility compared with control counterparts. Transcriptome analysis identified that genes associated with the endoplasmic reticulum (ER) function were dysregulated in Ufl1-deficient hearts. Biochemical analysis revealed that excessive ER stress preceded and deteriorated along with the development of cardiomyopathy in Ufl1-deficient hearts. Mechanistically, Ufl1 depletion impaired (PKR-like ER-resident kinase) signaling and aggravated cardiomyocyte cell death after ER stress. Administration of the chemical ER chaperone tauroursodeoxycholic acid to Ufl1-deficient mice alleviated ER stress and attenuated pressure overload-induced cardiac dysfunction. Our results advance a novel concept that the Ufm1 system is essential for cardiac homeostasis through regulation of ER function and that upregulation of myocardial Ufl1 could be protective against heart failure.

Abstract 530: The Ufm1 Specific Ligase 1 Regulates Endoplasmic Reticulum Homeostasis and Protects Against Heart Failure

Protein modifications by ubiquitin and ubiquitin-like proteins are emerging as an important regulatory mechanism of protein function and involved in various cellular processes. Ufm1 (ubiquitin-fold modifier 1) is a novel ubiquitin-like protein that modifies protein targets in a UBA5 (E1) - Ufc1 (E2) - Ufl1 (E3) dependent manner. While defects in ubiquitin- and SUMO-modification have been shown to provoke cardiac remodeling and dysfunction, the importance of Ufm1 modification has not been explored in the normal and stressed heart. Western blotting revealed that Ufm1 specific ligase 1 (Ufl1), an enzyme essential for Ufm1 modification, was increased in hypertrophic mouse hearts but reduced in the failing hearts of patients with dilated cardiomyopathy. To determine the functional role of Ufl1 in the heart, we generated a cardiac-specific knockout mouse and showed that Ufl1-deficient mice developed age-dependent cardiomyopathy and heart failure, as indicated by elevated cardiac fetal gene expression, increased fibrosis and impaired cardiac contractility. When challenged with pressure overload, Ufl1-deficient hearts exhibited remarkably greater hypertrophy, exacerbated fibrosis and worsened cardiac contractility compared with control counterparts. Transcriptome analysis identified that genes associated with the endoplasmic reticulum (ER) function were dysregulated in Ufl1-deficient hearts. Biochemical analysis revealed that excessive ER stress preceded and deteriorated along with the development of cardiomyopathy in Ufl1-deficient hearts. Mechanistically, Ufl1 depletion impaired PERK signaling and aggravated cardiomyocyte cell death following ER stress. Administration of the chemical ER chaperone tauroursodeoxycholic acid to Ufl1-deficient mice alleviated ER stress and attenuated pressure overload-induced cardiac dysfunction. Our results advance a novel concept that the Ufm1 system is essential for cardiac homeostasis through regulation of ER function and that upregulation of myocardial Ufl1 could be protective against heart failure.

Paroxetine-Mediated GRK2 Inhibition Reverses Cardiac Dysfunction and Remodeling After Myocardial Infarction

Background: Natriuretic peptides ProANP and Pro BNP are translated as biologically-inactive pro-hormones that require proteolytic cleavage into biologically active carboxyl-terminal fragments by the serine protease Corin. Activated ANP and BNP bind to GC-A or natriuretic peptide receptor A (NPRA), which increases intracellular second messenger cGMP, and further stimulates three known cGMP effector molecules: cGMP-dependent protein kinases (PKGs), cGMP-dependent phosphodiesterases (PDEs), and cGMP-dependent ion channels. This produces anti-hypertension, anti-hypertrophy and anti-fibrosis effects. The two linked SNP mutations in Corin gene are associated with risk for hypertension and left-ventricular hypertrophy in Blacks. Novel drugs are needed to target this pathway to improve cardiac functions that impaired by the genetic defects and complications caused by many types of heart failure. Methods and Results: We tested a NPR-A agonist PL3994, synthetic molecule made by Palatin Pharmaceutical Company for its potential therapeutic effects on heart failure and cardiac defects. By using our unique Tamoxifen inducible Corin cardiac specific knockout mouse model and Corin T623I/Q636P knockin mouse model, we implant osmotic pumps with PL3994 or PBS into the mice for four weeks while simultaneously constricted mouse aorta artery by TAC by microsurgery. We recorded cardiac function changes by blood pressure, ECHO, and at end stage measured gene expression profiles, hypertrophy, fibrosis and natriuretic pathway molecule: cGMP, cGMP dependent kinases, ANP28/ProANP128, BNP45/proBNP95 in the explanted hearts and plasma. Results. As compared to WT controls, Corin knockout mice and Corin T623I/Q636P knockin mice showed much worse cardiac hypertrophy by ECHO and heart weight and much worse cardiac fibrosis by Trichrome staining. These phenotypes are also supported by the evidences of less signaling cGMP, less PKG kinase activity in the Corin knockout and knockin mice. However, compared to placebo PBS saline, NPR-A agonist PL3994 significantly improved cardiac conditions with less cardiac hypertrophy and fibrosis, also together with increased signaling cGMP, less PKG kinase activity. Conclusions: NPR-A agonist PL3994 is a potential therapeutic drug to treat patients with Corin genetic defects and patients of heart failure caused by malfunctions of natriuretic pathways.

Cofilin-2 Phosphorylation and Sequestration in Myocardial Aggregates: Novel Pathogenetic Mechanisms for Idiopathic Dilated Cardiomyopathy

BackgroundRecently, tangles and plaque-like aggregates have been identified in certain cases of dilated cardiomyopathy (DCM), traditionally labeled idiopathic (iDCM), where there is no specific diagnostic test or targeted therapy. This suggests a potential underlying cause for some of the iDCM cases. ObjectivesThis study sought to identify the make-up of myocardial aggregates to understand the molecular mechanisms of these cases of DCM; this strategy has been central to understanding Alzheimer’s disease. MethodsAggregates were extracted from human iDCM samples with high congophilic reactivity (an indication of plaque presence), and the findings were validated in a larger cohort of samples. We tested the expression, distribution, and activity of cofilin in human tissue and generated a cardiac-specific knockout mouse model to investigate the functional impact of the human findings. We also modeled cofilin inactivity in vitro by using pharmacological and genetic gain- and loss-of-function approaches. ResultsAggregates in human myocardium were enriched for cofilin-2, an actin-depolymerizing protein known to participate in neurodegenerative diseases and nemaline myopathy. Cofilin-2 was predominantly phosphorylated, rendering it inactive. Cardiac-specific haploinsufficiency of cofilin-2 in mice recapitulated the human disease’s morphological, functional, and structural phenotype. Pharmacological stimulation of cofilin-2 phosphorylation and genetic overexpression of the phosphomimetic protein promoted the accumulation of “stress-like” fibers and severely impaired cardiomyocyte contractility. ConclusionsOur study provides the first biochemical characterization of prefibrillar myocardial aggregates in humans and the first report to link cofilin-2 to cardiomyopathy. The findings suggest a common pathogenetic mechanism connecting certain iDCMs and other chronic degenerative diseases, laying the groundwork for new therapeutic strategies.