Effects Of Parathyroid Hormone And Salt Interaction On Cardiac Metabolomics And Transcriptomics In Rats

Objective:To elucidate the effects of parathyroid hormone and salt interaction on rat myocardium morphology,metabolomics,and transcriptomics.To clarify the effects of PTH and salt intervention on mRNAs and metabolites in rat myocardium and compare the expression of differential mRNAs and metabolites in rat myocardium after different levels of salt intake,PTH,and combined intervention,and perform bioinformatic functional analysis of differential genes and metabolites.Methods:（1）Eighteen 8-week-old male Sprague Dawley rats were selected and randomly divided into the sham-operated group,PTH group,low-salt group（0.6%NaCl）,high-salt group（8%NaCl）,PTH+low-salt group（0.6%NaCl）,PTH+high-salt group（8%NaCl）,with three SD rats in each group.Throughout the feeding process,the growth environment was consistent,and all operations were in accordance with the ethical requirements of animal experiments at Dali University.（2）A capsule osmotic pressure pump was surgically implanted,and rat recombinant parathyroid hormone（1-34）was pumped at a dose of（2 pmol/kg.h）using an ALzet micropump in the PTH,PTH+low salt,and PTH+high salt groups,and equal amounts of saline were pumped in the sham-operated,low salt and high salt groups for a duration of 2 weeks with the same pumping rate.Gavage:The shamoperated and PTH groups were gavaged with medical sterile injection water,and the remaining groups were gavaged with different concentrations of NaCl（10 mg/kg/d）for two weeks according to the grouping.（3）The myocardial tissue of the left ventricle of each group of rats was taken and used for hematoxylin-eosin staining,transcriptomics,and metabolomics analysis,respectively;myocardial transcriptomics and metabolomics were detected and analyzed by Shanghai Biotec Biological Co.Transcriptomics sequencing technology analyzed differentially expressed genes after comparing among six groups,and GO analysis and enrichment analysis were performed for differentially expressed genes;metabolomics enriched differential metabolites after comparing among six groups;combined transcriptomics and metabolomics analyzed differential genes and metabolites after comparing among six groups,and identified key signaling pathways among them.Results:（1）Hematoxylin-eosin staining of myocardial tissues of rats in all groups revealed that myocardial cells were mildly hypertrophied with a little inflammatory cell infiltration around the myocardium when high salt was intervened with PTH alone compared with the sham-operated and low salt groups.Compared with the high salt and PTH interventions alone,the myocardial cells of rats under the combined PTH and high salt interventions were significantly hypertrophied,and myocardial cells were lysed and necrotic,accompanied by inflammatory cell infiltration.（2）Transcriptomic analysis revealed that fatty acid metabolism was reduced,and gluconeogenesis and amino acid metabolism were active in the rat myocardium after the high salt intervention.This led to an impairment of mitochondrial function in the myocardium,resulting in the development of myocardial oxidative stress.The differential genes in the high salt group,after comparison with the sham-operated and low salt groups,were mainly involved in the control of energy metabolic processes such as myocardial pyruvate metabolism,propionate metabolism,2-oxocarboxylic acid metabolism,glycolysis/gluconeogenesis,glyoxylate,and dicarboxylic acid metabolism,amino acid biosynthesis,starch,and sucrose metabolism,and glycerolipid metabolism;as well as upregulation of oxidative phosphorylation,myocardial contraction,and citric acid cycle processes within the myocardium;after PTH intervention,myocardial amino acid metabolism and glucose metabolism are active,and mitochondrial function is also affected,and oxidative stress is induced.The differential genes were mainly involved in the control of myocardial amino acid metabolisms such as valine/cysteine and methionine,pyruvate metabolism,carbon metabolism,cholesterol metabolism,and other energy metabolic processes,and the upregulation of myocardial oxidative phosphorylation,citric acid cycle,myocardial contraction process,FoxO signaling pathway,thyroid hormone signaling pathway,AGE-RAGE signaling pathway,ECM-receptor interaction,phospholipase D signaling pathway,PPAR signaling pathway,and p53 signaling pathway were activated in the myocardium.The combined intervention of high salt and PTH causes changes in energy metabolism,oxidative stress,and signaling pathways mentioned above and activates the HIF-1 signaling pathway in the myocardium.（3）Metabolomic analysis revealed that both high salt and PTH interventions alone and in concert for two weeks led to significant alterations in intra-myocardial metabolism in rats,and these alterations involved multiple metabolic pathways such as organic acid,amino acid,fatty acid,and purine metabolism.The metabolites that mainly affect intra-myocardial glycolysis or gluconeogenesis,pentose phosphate pathway,citric acid cycle,and amino acid metabolic processes;the intra-myocardial metabolic reprogramming involves mitochondria in the regulation of metabolic processes and active mitochondrial function in the non-physiological state（altered mitochondrial function has been verified at the transcriptomic level）,causing the onset of oxidative stress in the myocardium;the differential metabolites are also involved in intra-myocardial.The process of metabolic reprogramming in the myocardium is more pronounced with the combined intervention of PTH and high salt.（4）Metabolomic analysis of combined transcriptomics revealed that metabolism within the myocardium is reprogrammed after high salt intervention and leads to downregulation of metabolites important to the myocardium,such as ADP and pyruvate,and regulation of processes such as oxidative phosphorylation and citric acid cycle within the myocardium,and activation of the MAPK signaling pathway and Rap1 signaling pathway,AMPK/cAMP signaling pathway,PI3K-Akt signaling.The metabolism in the myocardium was also reprogrammed after PTH intervention,and metabolites such as D-glucose,pyruvate,and pyrophosphate were downregulated in the myocardium.Differential metabolites and genes were involved in regulating the oxidative phosphorylation and citric acid cycle in the myocardium,causing oxidative stress in the myocardium and activating the HIF-1 signaling pathway in the myocardium.Myocardial metabolic reprogramming was more pronounced with the combined intervention of high salt and PTH,and signaling pathways such as the AMPK signaling pathway and HIF-1 were also activated in the myocardium.Conclusion:Both high salt and PTH interventions cause reprogramming of myocardial metabolism,including fatty acid metabolism,amino acid metabolism,and glycolytic processes;mitochondrial function and oxidative phosphorylation processes are affected in the myocardium;myocardial pathology reveals cardiomyocyte hypertrophy with inflammatory cell infiltration,and Rap1,MAPK,PI3K-Akt,AMPK,cAMP,and HIF-1 signaling pathways were activated in the myocardium.In addition to the above metabolic and signaling pathway alterations,the combined intervention of high salt and PTH can lead to lysis necrosis of cardiac myocytes.