LncRNA LncHrt Preserves Cardiac Metabolic Homeostasis And Heart Function Post MI

Background:Ischemic heart disease like myocardial infarction is the leading cause of mortality and morbidity worldwide.Due to acute coronary artery blockage,myocardial infarction causes cardiac pump dysfunction,leading to ischemic cardiomyopathy with damaged myocardium tissue,massive fibrotic scar organization,cardiac contractile dysfunction and pathological cardiac dilation,as well as cardiac remodeling.Cardiac remodeling is not restricted to the region of infarcted zone but also affect the intact myocardium in remote area,leading to eccentric pathological hypertrophy accompanied by metabolic remodeling characterized by decreased fatty acid oxidation and increased glycolysis which decreases the ATP content.Reduced energy reserve contribute to increased susceptibility of MI heart and drive the pathogenesis and progression of heart failure.Optimizing cardiac enegy metabolic homeostasis has been an effective therapeutic intervention in ischemic heart disease.But the role of LncRNA in cardiac metabolic regulation is rarely reported.Using microarray-based transcriptome profiling on the mouse hearts post myocardial infarction(MI)or sham control,we identify a cardiomyocytes enriched LncRNA named LncHrt,Overexpression of LncHrt post myocardial infarction improved cardiac function,playing a protective role in preserving cardiac metabolic homeostasis and attenuating adverse cardiac remodeling.Objective:We sought to investigate the role of LncRNA LncHrt in cardiac homeostasis and its protective effect on heart energy balance modulation and cardiac function post myocardial infarction,elucidating the regulatory mechanisms of LncHrt in cardiac metabolic homeostasis.Methods:Myocardial infarction(MI)in mouse model were established by ligating the left anterior descending(LAD)coronary artery permanently.Using microarray-based transcriptome profiling on the heart tissues post myocardial infarction we identified down-regulated LncRNAs both in MI-3d and MI-14 d significantly.Then combined analysis of the gene differencially expesssion,tissue distribution and heart enrichment,we narrow down to LncRNA AK034241,named LncHrt.We detect LncHrt gene expression in heart development,cardiac cell type distribution and subcellular location by qRT-PCR.We then performed 5’ and 3’ rapid amplification of cDNA ends(RACE)using RNAs isolated from adult mouse heart and achieved the full length of LncHrt.We then dissected the conservation of LncHrt in UCSC Genome Browser(GRCm38/mm10)and predicted LncHrt secondary structure by RegRNA Server.We validated the expression of human homology of mouse LncHrt was decreased in patients with dilated cardiomyopathy(DCM)which was in accordance with the expression pattern of mouse LncHrt in MI model.We knocked down LncHrt expression level in vivo by AAV9-shRNA(short hairpin RNA)against LncHrt to see its role in cardiac homeostasis.We employed AAV9-mediated delivery system to overexpress LncHrt in vivo,in which the cardiac-specific cTNT promoter has been incorporated to achieve cardiomyocytes specific overexpression of LncHrt to investigate its protective role in the cardiac metabolic homeostasis and heart function in the pathological progression of MI.Using Sirius red and Fast green staining to examine cardiac fibrosis and scar size post MI.Using unbiased genome-wide RNA sequencing(RNA-seq)on ventricular tissues to gain a deep understanding of the molecular mechanisms underlying LncHrt-mediated cardiac protection following MI,Database for Annotation,Visualization and Integrated Discovery(DAVID)tools were used to analyze functional annotation of differentially expressed genes.we performed comparative analyses of transcriptome signatures from both control and MI hearts with or without LncHrt overexpression using Gene Set Enrichment Analysis(GSEA)Gene Set Enrichment Analysis(GSEA)to further validate LncHrt regulation in cardiac metabolism.We assessed mitochondrial respiration by measuring oxygen consumption rate(OCR)in permeabilized mouse myofibers using high resolution respirometry following substrate-uncoupler-inhibitor-titration(SUIT)protocols,ADP-stimulated oxygen flux was determined under coupled conditions to estimate the mitochondrial function.RNA pull-down assay coupled with mass spectrometry identified LncHrt-interacting protein SIRT2,RNA immunoprecipitation(RIP)assay further confirmed the interaction between LncHrt and SIRT2 endogenously.Western blot assay was used to detect LncHrt regulated LKB1-AMPK signaling pathway,Co-immunoprecipitation validated LncHrt target SIRT2 interaction with CDK5,and Immunoprecipitated LKB1 was used to research SIRT2 regulation on LKB1 acytylation modification.Results:Part Ⅰ:LncRNAs screening1.LncRNAs screening we performed microarray-based transcriptome profiling on mouse hearts 3 and 14 days after myocardial infarction(MI)and compared these to sham-operated control mice.Using combined analyses,we identified significantly downregulated LncRNA in both MI-3d and MI-14 d time points and further validated a novel LncRNA which was specially enriched in the heart and highly expressed in cardiomyocytes,expressed in both cytoplasm and nuclear,named LncHrt.LncHrt was progressively elevated 7 days after birth and then dramatically increased until adulthood with a peak value in P21d;indicating LncHrt may be essential for adult heart homeostasis and may be related with substrte metabolism.2.LncRNA LncHrt cloning and validation We performed 5’ and 3’ rapid amplification of cDNA ends(RACE)using RNA isolated from adult mouse hearts and achieved the full length of LncHrt.using the Coding Potential Calculator revealed that the LncHrt transcript has a very low coding-potential score,similar to Chaer and Hotair,two well-known lncRNAs.Indicating the LncHrt transcript is a true non-coding RNA.3.LncHrt conservation and its expression human dialated cardiomyopathy The genome locus of LncHrt in humans is also in chromosome 14,while the LncHrt sequence is highly-conserved across multiple species in exon 1 as well as the 5’-end and3’-end of exon 2.The predicted LncHrt secondary structure using the RegRNA Server revealed that LncHrt has stem-loop structures with relatively high base-pairing potential.Consistent with the finding that mouse LncHrt was downregulated in the heart after myocardial infarction,we found that the expression of the human homolog of mouse LncHrt was dramatically decreased in patients with dilated cardiomyopathy(DCM)suggesting that this lncRNA may play in important role in the regulation of heart disease.Part Ⅱ:Knock-down of LncHrt impairs cardiac homeostasis1.To define the in vivo function of LncHrt in the heart,we knocked-down LncHrt expression using AAV9-shRNA(short hairpin RNA)directed against LncHrt in neonatal mice.LncHrt inhibition resulted in enlarged heart,the heart weight to body weight ratio(HW/BW)was also significantly increased in these mice.Echocardiography revealed that AAV-LncHrt KD impaired left ventricular systolic heart function and decreased fractional shortening.In particular,AAV-LncHrt KD hearts exhibited a dilated left ventricular chamber,and a reduced left ventricular posterior wall thickness.2.We observed markedly increased collagen deposition which indicate cardiac fibrosis in AAV-LncHrt KD hearts histologically,validated cardiomyocytes pathological hypertrophy with molecular marker analyses demonstrated a significantly increase in the expression of cardiomyopathy marker genes such as Nppa,Nppb,and Mhy7 after LncHrt knock-down which supports our observation that knock-down of LncHrt adversely affects cardiac function.Part Ⅲ:LncHrt overexpression protects the heart from myocardial infarction1.We employed AAV9-mediated delivery system to overexpress LncHrt in vivo using the cTNT promoter to achieve cardiomyocyte-specific overexpression of LncHrt.Remarkably,overexpression of LncHrt improved cardiac morphology and significantly reduced the HW/BW ratio in response to MI compared to the control group.More importantly,AAV-LncHrt mice exhibited preserved cardiac function after MI,as determined by echocardiography that showed improved fractional shortening,maintenance of left ventricular posterior wall thickness,and reduced cardiac chamber internal diameter.2.Histological analyses by sirius red and fast green staining revealed that overexpression of LncHrt in the heart substantially reduced infarct fibrosis following MI when compared to controls.In addition,cardiomyocyte size was markedly decreased in AAV-LncHrt treated hearts post-MI,indicating reduced cardiac pathological remodeling.Molecular marker analyses demonstrated a significant decrease in the expression of the hypertrophic and cardiomyopathy marker genes Nppa,Nppb and Myh7 in AAV-LncHrt treated hearts,further indicating LncHrt plays a protective role in cardiac function and attenuates adverse cardiac remodeling in response to MI.Part Ⅳ:LncHrt overexpression rescues the transcriptome in infarcted hearts1.We performed genome-wide RNA sequencing on ventricular tissue from AAV9-mediated LncHrt overexpression and control hearts 7 days post-MI.We analyzed the functional annotation of these differentially expressed genes and confirmed that "metabolic processes" and "cardiac muscle contraction" are enriched among upregulated genes while "immune system process","inflammatory response",and "collagen fibril organization" are enriched in downregulated genes.2.We performed comparative analyses of transcriptome signatures from both control and MI hearts with or without LncHrt overexpression.Gene Set Enrichment Analysis(GSEA)demonstrated that MI-induced down-regulation of extensive metabolic processes such as "oxidative phosphorylation","fatty acid metabolism",and "glycolysis" were completely restored upon LncHrt overexpression compared to control post-MI.Cardiac pathological pathways like "ECM receptor interaction" and "TGF beta signaling pathway" were repressed by LncHrt in MI hearts and "cardiac muscle contraction" was increased.These analyses support the notion that LncHrt plays an important role in cardiac metabolism and muscle contraction following MI.3.To confirm these results experimentally,we collected heart samples from an independent set of animals and performed qRT-PCR analysis for differentiallyexpressed signature genes.Our results validated the changes described above in multiple pathways including "carbon metabolism","TCA cycle","fatty acid metabolism","glycolysis","fatty acid degradation","pyruvate metabolism",and "cardiac muscle contraction".In contrast,knock-down of LncHrt resulted in an opposite effect on metabolic gene expression Part Ⅴ:LncHrt improved cardiac metabolic homeostasis post-MI1.In the remote ischemic zone,LncHrt promote respiratory stimulation of the FAOpathway,complex Ⅰ,complex Ⅱ and complex Ⅵ OXPHOS capacity and ATP production.2.LncHrt enhanced protein levels of HK1,PGK1,CS as well as IDH2 which are both key glycolytic enzymes and energy production enzymes in the infarct of hearts under hypoxic conditions.indicating LncHrt increased glucose utilization during the TCA cycle and mitochondrial oxidation under hypoxic conditions post-MI.3.While knock-down of LncHrt impaired mitochondrial respiration,fatty acid oxidation and oxidative phosphorylation.Part Ⅵ:LncHrt activates the LKB1-AMPK signaling pathway via SIRT21.LncHrt didn’t regulate the expression of its neighboring gene,Klhl33.We overexpressed LncHrt to assess the transcriptional impact on Klhl33 in vivo and in vitro.qRT-PCR analysis showed that LncHrt overexpression had no effect on Klhl33 gene expression in vivo before or after MI and overexpression of LncHrt did not affect Klhl33 levels in vitro.2.RNA pull-down assay coupled with mass spectrometry identified LncHrt interating protein SIRT2,RNA immunoprecipitation further confirmed the endogenous interaction between LncHrt and SIRT2.LncHrt interacts with SIRT2 to regulate its function by modulating LKB1-AMPK signaling in the heart.Part Ⅶ:LncHrt preserves SIRT2 activity by blocking CDK5 inhibition1.LncHrt did not affect Sirt2 transcript level or protein expression since Sirt2 was not changed in the LncHrt gain-of-function or loss-of-function models.2.LncHrt exhibited cardiac metabolic protection by interfering the CDK5 inhibition on the catalytic activity of SIRT2,leading to LKB1-AMPK signal activation.Conclusions:We identified a novel cardiomyocyte-enriched lncRNA called LncHrt,which was sharply down-regulated in ischemic heart disease.By inhibiting LncHrt expression we find LncHrt plays an essential role in maintaining normal cardiac function.while AAVbased LncHrt overexpression protects the heart from MI as demonstrated by improved contractile function,preserved cardiac metabolic homeostasis,and attenuated adverse remodeling responses.mechanistically,LncHrt interacts with SIRT2 to preserve SIRT2 deacetylase activity by interfering with the CDK5 and SIRT2 interaction.This increases downstream LKB1-AMPK kinase signaling,which ameliorates functional and metabolic deficits.