#METABOLOMICS WORKBENCH RobertHume_20251022_150443 DATATRACK_ID:6589 STUDY_ID:ST004323 ANALYSIS_ID:AN007203 PROJECT_ID:PR002737 VERSION 1 CREATED_ON October 29, 2025, 10:57 pm #PROJECT PR:PROJECT_TITLE Human Hearts intrinsically increase cardiomyocyte mitosis following myocardial PR:PROJECT_TITLE infarction PR:PROJECT_TYPE Metabolomics PR:PROJECT_SUMMARY Background: Myocardial infarction (MI) is a leading cause of death worldwide and PR:PROJECT_SUMMARY can eliminate up to a third of the cardiomyocytes (CMs) within the human heart. PR:PROJECT_SUMMARY Although CMs undergo mitosis during early development, most CMs cease cell PR:PROJECT_SUMMARY cycling soon after birth. In contrast, rodent MI models have shown that CMs PR:PROJECT_SUMMARY increase mitosis in response to ischemia, however this has not been shown in PR:PROJECT_SUMMARY humans. Methods: Using a unique pre-mortem post-MI human heart, immunostaining, PR:PROJECT_SUMMARY bulk RNA sequencing, proteomics, metabolomics, single nucleus RNA sequencing and PR:PROJECT_SUMMARY a novel post-MI human biopsy method, we investigated human CM mitosis post-MI. PR:PROJECT_SUMMARY Results: We show that adult human CMs exhibit increased mitosis and cytokinesis PR:PROJECT_SUMMARY in response to ischemia. Conclusions: Future development of therapeutics to PR:PROJECT_SUMMARY enhance this intrinsic mitotic potential could lead to new treatments that PR:PROJECT_SUMMARY reverse heart failure via cardiac regeneration. PR:INSTITUTE University of Sydney PR:DEPARTMENT Charles Perkins Centre PR:LABORATORY Centre for Heart Failure and Diseases of the Aorta PR:LAST_NAME Robert PR:FIRST_NAME Hume PR:ADDRESS John Hopkins Dr, Camperdown, NSW 2050, Australia PR:EMAIL robert.hume@sydney.edu.au PR:PHONE 0434848772 PR:PUBLICATIONS Circulation Research (in print) #STUDY ST:STUDY_TITLE Human Hearts intrinsically increase cardiomyocyte mitosis following myocardial ST:STUDY_TITLE infarction ST:STUDY_SUMMARY Background: Myocardial infarction (MI) is a leading cause of death worldwide and ST:STUDY_SUMMARY can eliminate up to a third of the cardiomyocytes (CMs) within the human heart. ST:STUDY_SUMMARY Although CMs undergo mitosis during early development, most CMs cease cell ST:STUDY_SUMMARY cycling soon after birth. In contrast, rodent MI models have shown that CMs ST:STUDY_SUMMARY increase mitosis in response to ischemia, however this has not been shown in ST:STUDY_SUMMARY humans. Methods: Using a unique pre-mortem post-MI human heart, immunostaining, ST:STUDY_SUMMARY bulk RNA sequencing, proteomics, metabolomics, single nucleus RNA sequencing and ST:STUDY_SUMMARY a novel post-MI human biopsy method, we investigated human CM mitosis post-MI. ST:STUDY_SUMMARY Results: We show that adult human CMs exhibit increased mitosis and cytokinesis ST:STUDY_SUMMARY in response to ischemia. Conclusions: Future development of therapeutics to ST:STUDY_SUMMARY enhance this intrinsic mitotic potential could lead to new treatments that ST:STUDY_SUMMARY reverse heart failure via cardiac regeneration. This uploaded dataset relates to ST:STUDY_SUMMARY the metabolomics (HILIC and AMIDE) performed on the unique infarcted human heart ST:STUDY_SUMMARY (5 days post-myocardial infarction) as compared to healthy donor hearts. Infarct ST:STUDY_SUMMARY samples consisted of non-ischemic right atrium (MI RA, technical replicates), ST:STUDY_SUMMARY partially ischemic right ventricle (MI RV, technical replicates), ischemic left ST:STUDY_SUMMARY ventricle (MI LV, technical replicates) and non-ischemic left ventricle donor ST:STUDY_SUMMARY tissue (Donor LV, biological replicates). ST:INSTITUTE University of Sydney ST:DEPARTMENT Charles Perkins Centre ST:LAST_NAME Hume ST:FIRST_NAME Robert ST:ADDRESS John Hopkins Dr ST:EMAIL robert.hume@sydney.edu.au ST:PHONE 0434848772 ST:DISEASE_ASSOCIATION Donor left ventricle or infarct (MI) left ventricle, right ventricle or right ST:DISEASE_ASSOCIATION atrium #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_LV10 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=1; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_LV10.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_LV14 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=2; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_LV14.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_LV18 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=3; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_LV18.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_LV26 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=4; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_LV26.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_LV28 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=5; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_LV28.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_RA1_02 Sample source:Infarcted human heart right atrium | Tissue Type:RA | Group:Infarct (non-ischemic) Technical Replicates=1; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_RA1_02.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_RA2 Sample source:Infarcted human heart right atrium | Tissue Type:RA | Group:Infarct (non-ischemic) Technical Replicates=2; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_RA2.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_RV1 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=1; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_RV1.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_RV2 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=2; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_RV2.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_RV3 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=3; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_RV3.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_RV4 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=4; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_RV4.wiff SUBJECT_SAMPLE_FACTORS 6.004 HILIC_6.004ROB_RV5 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=5; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_6.004ROB_RV5.wiff SUBJECT_SAMPLE_FACTORS 3.145 HILIC_CASS3.145LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=1; RAW_FILE_NAME(File Name)=HILIC_CASS3.145LV.wiff SUBJECT_SAMPLE_FACTORS 6.048 HILIC_CASS6.048LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=2; RAW_FILE_NAME(File Name)=HILIC_CASS6.048LV.wiff SUBJECT_SAMPLE_FACTORS 6.056 HILIC_CASS6.056LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=3; RAW_FILE_NAME(File Name)=HILIC_CASS6.056LV.wiff SUBJECT_SAMPLE_FACTORS 7.004 HILIC_CASS7.004LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=4; RAW_FILE_NAME(File Name)=HILIC_CASS7.004LV.wiff SUBJECT_SAMPLE_FACTORS 7.08 HILIC_CASS7.080LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=5; RAW_FILE_NAME(File Name)=HILIC_CASS7.080LV.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_LV10 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=1; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_LV10.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_LV14 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=2; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_LV14.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_LV18 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=3; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_LV18.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_LV26 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=4; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_LV26.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_LV28 Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Technical Replicates=5; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_LV28.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_RA1_02 Sample source:Infarcted human heart right atrium | Tissue Type:RA | Group:Infarct (non-ischemic) Technical Replicates=1; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_RA1_02.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_RA2 Sample source:Infarcted human heart right atrium | Tissue Type:RA | Group:Infarct (non-ischemic) Technical Replicates=2; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_RA2.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_RV1 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=1; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_RV1.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_RV2 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=2; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_RV2.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_RV3 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=3; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_RV3.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_RV4 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=4; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_RV4.wiff SUBJECT_SAMPLE_FACTORS 6.004 AMIDE_6.004ROB_RV5 Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Technical Replicates=5; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_6.004ROB_RV5.wiff SUBJECT_SAMPLE_FACTORS 3.145 AMIDE_CASS3.145LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=1; RAW_FILE_NAME(File Name)=AMIDE_CASS3.145LV.wiff SUBJECT_SAMPLE_FACTORS 6.048 AMIDE_CASS6.048LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=2; RAW_FILE_NAME(File Name)=AMIDE_CASS6.048LV.wiff SUBJECT_SAMPLE_FACTORS 6.056 AMIDE_CASS6.056LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=3; RAW_FILE_NAME(File Name)=AMIDE_CASS6.056LV.wiff SUBJECT_SAMPLE_FACTORS 7.004 AMIDE_CASS7.004LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=4; RAW_FILE_NAME(File Name)=AMIDE_CASS7.004LV.wiff SUBJECT_SAMPLE_FACTORS 7.08 AMIDE_CASS7.080LV Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Technical Replicates=1; Biological replicates=5; RAW_FILE_NAME(File Name)=AMIDE_CASS7.080LV.wiff #COLLECTION CO:COLLECTION_SUMMARY Human Heart tissue Donor hearts (Sydney Heart Bank) deemed suitable for heart CO:COLLECTION_SUMMARY transplantation but unable to be transplanted (for reasons including CO:COLLECTION_SUMMARY transportation logistics, immune incompatibility, and donor-recipient mismatch CO:COLLECTION_SUMMARY in size) were procured as previously described40. These are not post-mortem CO:COLLECTION_SUMMARY samples. These non-diseased donor heart samples were from patients with a CO:COLLECTION_SUMMARY non-cardiac cause of death and no significant co-morbidities or cardiac risk CO:COLLECTION_SUMMARY factors (hence why the hearts would otherwise have been used for transplant). CO:COLLECTION_SUMMARY All hearts (non-diseased donor or ischemic) underwent formal pathological CO:COLLECTION_SUMMARY examination by clinical anatomical pathology to confirm either normal CO:COLLECTION_SUMMARY histological architecture or ischemic heart disease respectively. LV samples CO:COLLECTION_SUMMARY were obtained immediately after harvest and snap frozen in liquid nitrogen (-196 CO:COLLECTION_SUMMARY °C). The study was approved by the Human Ethics Committee of The University of CO:COLLECTION_SUMMARY Sydney (USYD # 2021/122). Procurement of a unique infarcted human heart The CO:COLLECTION_SUMMARY unique infarcted human heart was acquired from a 48yo male patient, originally CO:COLLECTION_SUMMARY on the national donor list, who suffered a catastrophic acute myocardial CO:COLLECTION_SUMMARY infarction (MI) due to a blockage of the left anterior descending artery. This CO:COLLECTION_SUMMARY patient suffered a cardiac arrest and at the Royal Prince Alfred Hospital, CO:COLLECTION_SUMMARY Sydney, whereby a coronary angioplasty and stent insertion could not be CO:COLLECTION_SUMMARY achieved. The patient was then kept on life support for 5d post-MI, at which CO:COLLECTION_SUMMARY point no neurological activity was detected and the patient was declared CO:COLLECTION_SUMMARY braindead. As the heart could not be used for transplant purposes, the next of CO:COLLECTION_SUMMARY kin kindly agreed to organ donation for research. The heart was then collected CO:COLLECTION_SUMMARY pre-mortem and cryopreserved within 15mins i.e. fragments from the right atrium CO:COLLECTION_SUMMARY (MI RA), right ventricle (MI RV) and left ventricle (MI LV) were flash frozen CO:COLLECTION_SUMMARY and stored in liquid nitrogen, or fixed in 10% neutral buffered formalin (NBF, CO:COLLECTION_SUMMARY Sigma HTS012) overnight, before use. CO:SAMPLE_TYPE Heart CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY No treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were fragmented with metal bead and an extraction buffer of equal SP:SAMPLEPREP_SUMMARY methanol chloroform. Once tissue is fully lysed, chloroform and HPLC water is SP:SAMPLEPREP_SUMMARY added before centrifugation. Two layers are aliquoted and separated which was SP:SAMPLEPREP_SUMMARY ran in the mass spectrometer. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Sciex 6500+ CH:COLUMN_NAME Waters XBridge AMIDE 100 x 2.1 mm, 3.5 um CH:SOLVENT_A 5% acetonitrile, 95% water containing 10 mM ammonium acetate with pH at 9 CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 85%B and decreased after 8min to 35% and continue to decrease to 2% at 9min and CH:FLOW_GRADIENT maintained for 2min before ramping back to 85% within 1min CH:FLOW_RATE 0.25ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6500+ QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The extracted samples were analysed on Sciex 6500 (LC-MS/MS systems) with MS:MS_COMMENTS parameter settings as following: curtain gas 70 psi; ion source gas 1 at 30 psi; MS:MS_COMMENTS gas 2 at 70 psi; source temperature at 350 ˚C; ion spray voltage was set at MS:MS_COMMENTS 4500 V at negative mode and 3500 v at positive mode. All MRM transitions, MS:MS_COMMENTS collision energy, and declustering potentials were optimised using authentic MS:MS_COMMENTS chemical standards. Scheduled MRMs were based on retention time of each MS:MS_COMMENTS metabolite and on average of 10-30 ms dwell time were used to ensure sufficient MS:MS_COMMENTS data points across each peak.    #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AUC MS_METABOLITE_DATA_START Samples AMIDE_6.004ROB_LV10 AMIDE_6.004ROB_LV14 AMIDE_6.004ROB_LV18 AMIDE_6.004ROB_LV26 AMIDE_6.004ROB_LV28 AMIDE_6.004ROB_RA2 AMIDE_6.004ROB_RV1 AMIDE_6.004ROB_RV2 AMIDE_6.004ROB_RV3 AMIDE_6.004ROB_RV4 AMIDE_6.004ROB_RV5 AMIDE_CASS3.145LV AMIDE_CASS6.048LV AMIDE_CASS6.056LV AMIDE_CASS7.004LV AMIDE_CASS7.080LV AMIDE_6.004ROB_RA1_02 Factors Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Sample source:Infarcted human heart left ventricle | Tissue Type:LV | Group:Infarct Sample source:Infarcted human heart right atrium | Tissue Type:RA | Group:Infarct (non-ischemic) Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Sample source:Infarcted human heart right ventricle | Tissue Type:RV | Group:Infarct (semi-ischemic) Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Sample source:Donor human heart left ventricle | Tissue Type:LV | Group:Donor Sample source:Infarcted human heart right atrium | Tissue Type:RA | Group:Infarct (non-ischemic) Acetoacetate 3047665.561 2853215.994 2431951.214 2864678.09 2725876.714 3042506.749 2390531.556 2941454.24 2569339.524 3298418.41 2392280.186 2335063.88 2289774.839 2453379.976 1655557.299 2545111.502 3491467.215 Pyruvate.1 97199.65165 73710.75215 85329.68965 105496.3033 53934.61343 55124.64076 57360.68849 82594.68259 71310.04759 102877.6311 101738.7764 115032.8291 62042.38032 63539.95249 43229.5129 104197.5084 70813.71044 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name HMDB KEGG Acetoacetate HMDB0000060 C00164 Pyruvate.1 NA C00022 METABOLITES_END #END