Mechanisms Of Improvement In Sodium-Glucose Transporter 2 Inhibitors For Salt-Sensitive Hypertension And Cardiovascular Damage

Background and Aims:Hypertension is one of the most common chronic diseases,with over one billion people worldwide suffering from hypertension.Hypertension is a major risk to human health because it could damage the target organs such as the heart,brain and kidneys,and even lead to complications such as myocardial infarction,heart failure,cerebral haemorrhage,cerebral infarction and renal failure.Salt is an important environmental factor in primary hypertension.Numerous studies have shown that salt intake is directly related to the development of cardiovascular disease.The prevalence of hypertension in China is about 20%,of which salt-sensitive hypertension accounts for 50%.The mechanisms of salt-sensitive hypertension are mainly through sodium transport mechanisms,sympathetic activation,renal sodium excretion impairment and inflammatory response,renin-angiotensin-aldosterone system（RAAS）,endothelial impairment,and endocrine mechanisms.Sodium restriction is an effective intervention,but has not been shown to significantly reduce sodium intake in the population.In addition,urinary sodium excretion promoted by diuretics may also cause electrolyte disturbances and increase cardiovascular risk.The choice of an alternative therapy that reduces the harmful effects of high salt intake is essential in the treatment of salt-sensitive hypertensionIn recent years,a new class of anti-diabetic drugs,SGLT2 inhibitors（SGLT2i）,including canagliflozin,dagliflozin and engramlizin have shown impressive renal and cardiovascular benefits through several clinical trials.Impressive renal and cardiovascular benefits of SGLT2i have been shown through several large clinical trials,reducing all-cause and cardiovascular mortality in diabetic and non-diabetic patients.The potential mechanisms for the cardiovascular benefits of SGLT2i are diverse and may involve a reduction in plasma volume,a reduction in blood glucose,an increase in urinary sodium excretion and a reduction in RAAS activation.All of these may contribute to lower blood pressure.However,it is unclear whether SGLT2i have a direct effect on vascular function and blood pressure.In addition,hypertension is a major pathogenesis of heart failure,which is the most important and serious target organ damage in salt-sensitive hypertension,with persistently high rates of disability and death and very high societal costs.Sodium restriction can also reduce blood pressure and improve the incidence of heart failure,but current salt restriction strategies are difficult to achieve and manage.SGLT2i have been clinically shown to significantly improve heart failure in both diabetic and non-diabetic patients.Clarifying whether SGLT2i could improve high salt-induced cardiac hypertrophy and its mechanism,and exploring the direct mechanism by which high salt causes cardiac hypertrophy,is essential to prevent the development of heart failure and salt-sensitive hypertension.Clinical and animal studies have shown that high salt diet is an important pathogenetic and environmental factor in myocardial hypertrophy independent of blood pressure,and the mechanism of interaction between salt and myocardial hypertrophy is currently unknown.The myocardium has a large number of mitochondria,and the adult cardiomyocytes obtain energy mainly from fatty acid oxidation,and mitochondrial dysfunction is an important cause of cardiac hypertrophy and heart failure.Previous studies have shown that the conversion of cardiomyocytes from fatty acid to glucose energy supply is an important feature of myocardial hypertrophy.However,recent studies have found that high-salt diet leads to increased glucose uptake by cardiomyocytes,even with acute intervention.Whether the high-salt diet affects myocardial hypertrophy and heart failure through glucose metabolism remains unclear.SIRT3 is a mitochondrial NAD+-dependent deacetylase that plays a key role in the development of myocardial hypertrophy.Previous studies have focused on ameliorating oxidative stress and mitochondrial permeability through SIRT3 to protect against myocardial hypertrophy and cell death.In our previous study,we observed a decrease in cardiac SIRT3expression in the mice fed with high salt diet.However,it is worth investigating further whether SGLT2 inhibitors improve SIRT3-dependent mitochondrial function and thereby ameliorate high salt-induced myocardial hypertrophy and heart failure.To validate the above hypothesis,this study was designed in two parts.The first part was to clarify the antihypertensive effect of SGLT2i on salt-sensitive hypertension and its mechanism,and to elucidate the role of TRPC3 in salt-sensitive hypertension.The second part was to investigate the protective effect of SGLT2i on high salt diet-induced myocardial hypertrophy and its mechanism,and to clarify the key roles of mitochondrial dysfunction and SIRT3 in high salt-induced myocardial hypertrophy.Materials and methods:This study was validated by in vivo animal experiments and in vitro cell culture experiments.First,in the animal part,salt-sensitive rats,salt-insensitive rats were fed with normal chow（general diet group）,8%Na Cl chow（high salt group）and high salt chow+canagliflozin（high salt+canagliflozin group）for 12 weeks.129J background wild-type（WT）,TRPC3 knockout(Trpc3^-/-)and myocardial SIRT3 gene conditional knockout(SIRT3^flox/floxmyh6^cre)models on a C57BL/6J background were given normal chow（general diet group）,8%Na Cl chow（high salt group）and high salt chow+canagliflozin（high salt+canagliflozin group）for 24 weeks.The isolated experiments were performed on mouse vascular smooth muscle cell（VSMCs）lines.1.Salt-sensitive rats and salt-insensitive rats were treated with normal diet,high salt diet and high salt+canagliflozin diet for 12 weeks,and tail blood pressure was monitored weekly by tail blood pressure measurements.At the end of the intervention,24-hour ambulatory blood pressure was measured using dynamic wireless implants.2.At the end of the intervention,small animal ultrasound was used to examine the cardiac structure and function of the mice.Biochemical parameters such as body weight,blood glucose,blood urine creatinine,urea nitrogen,24-hour urine volume,urine sodium and potassium were collected.Heart and aortic tissue was collected for proteomic sequencing,paraffin sectioning and subsequent protein analysis at the end of the study.3.Mesenteric arteries were isolated to detect changes in vascular function and to observe changes in vascular diastolic and systolic function in each group of rats.HE and immunofluorescence staining were performed to observe changes in vascular structure and the distribution of TRPC3 and NCX1 proteins.4.To clarify whether TRPC3 is a differential gene for salt-sensitive hypertension based on proteomics and Western-blot results.5.TRPC3 overexpression/silencing plasmids were used to transfect VSMCs and TRPC3knockout mice to verify the role of TRPC3 in high salt on cytoplasmic calcium and vasoconstriction.6.The specific mechanism of TRPC3 regulation of vasoconstriction was investigated by TRPC3 overexpression/silencing plasmid transfection of VSMCs and TRPC3 knockout mice using mesenteric artery function and cytoplasmic calcium levels.7.Western-blot assay was used to detect the effect of high salt and canagliflozin on the expression of proteins related to vasoconstriction and calcium-regulated signaling pathways（TRPC3,NCX1,Camk II,p-Camk II,MYPT-1,p-MYPT-1,MLC,p-MLC）in vascular smooth muscle cells.8.To detect the effects of high salt diet and canagliflozin on the mitochondrial respiratory chain oxidative phosphorylation function and fatty acid oxidation function in rat cardiomyocytes using the mitochondrial respiration instrument.9.As indicators of myocardial hypertrophy,section staining of HE and WGA and m RNA levels of ANP,BNP andβ-MHC were observed.10.To clarify whether SIRT3 is a differential gene for high salt-induced myocardial hypertrophy and an intervention target for canagliflozin based on proteomics and Western-blot results.11.Construct a myocardial SIRT3 knockout mouse model to validate the role of SIRT3in high salt-induced myocardial hypertrophy and the drug target of canagliflozin.12 Western-blot assay was performed to detect the effects of high salt and canagliflozin on the expression of cardiomyocyte metabolic pathway-related proteins and mitochondrial proteins（CPT1,PDHα,LDHα,MPC1 and SIRT3）.Results:1.High salt diet could increase blood pressure in salt-sensitive rats,but not in salt-insensitive rats.Canagliflozin could significantly reduce blood pressure in salt-sensitive rats,but not in salt-insensitive rats.2.Canagliflozin promoted urinary sodium excretion and improved renal function in salt-sensitive rats and salt-insensitive rats,and more importantly,improved vascular remodelling in salt-sensitive rats.3.Canagliflozin improved endothelium-dependent/endothelium-independent diastolic function in salt-sensitive and salt-insensitive rats,but canagliflozin significantly inhibited vasoconstriction in salt-sensitive rats.4.TRPC3 expression was significantly increased in salt-sensitive rats fed with high salt diet,and canagliflozin significantly decreased TRPC3 expression in salt-sensitive rats.5.The proteomic results showed that high salt mainly affected the protein expression of calcium-regulated and vasoconstriction-associated pathways in the vasculature,and that canagliflozin mainly inhibited the protein expression of calcium-regulated and vasoconstriction-associated pathways.6.Overexpression/silencing of the TRPC3 gene increased/decreased cytoplasmic free calcium levels in vascular smooth muscle cells,which affected the expression levels of vasoconstrictor pathway proteins such as p-MYPT-1 and p-MLC via Camk II phosphorylation.7.Knockout of the TRPC3 gene ameliorated high salt diet-induced blood pressure elevation and vascular remodelling,and its reason mainly attributed to improve the high salt-induced mesenteric artery contraction.8.High salt diet promotes increased cytoplasmic free calcium levels and vasoconstriction through the binding of TRPC3 to NCX1,whereas canagliflozin can reduce cytoplasmic free calcium levels and vasoconstriction by inhibiting TRPC3 binding to NCX1.9.Long-term high-salt diet induced myocardial hypertrophy in salt-sensitive rats.Both HE and WGA showed myocardial hypertrophy,and m RNA of heart failure markers such as ANP,BNP andβ-MHC were significantly increased.10.Long-term high-salt diet significantly impaired oxidative phosphorylation and fatty acid oxidation in the heart mitochondria of salt-sensitive rats,and canagliflozin significantly improved oxidative phosphorylation and fatty acid oxidation in the heart mitochondria of salt-sensitive rats.11.Proteomics results showed that high salt mainly affected mitochondrial metabolism and glucose metabolic pathway,in which SIRT3 was the differential gene.Canagliflozin mainly improved the mitochondrial metabolic pathway,in which SIRT3 was also the differential gene.Western-blot verified that SIRT3 mainly regulated CPT1,PDHα,LDHα,MPC1 and other mitochondrial metabolic pathway proteins to affect cardiac hypertrophy.12.Conditional knockdown of the myocardial SRIT3 gene exacerbated high salt-induced myocardial hypertrophy and attenuated the protective effect of canagliflozin.Conclusions:1.Canagliflozin significantly reduced blood pressure and vascular remodelling in salt-sensitive hypertensive rats,probably through inhibition of TRPC3-mediated cytoplasmic free calcium levels and vasoconstriction.2.High salt activated TRPC3 and NCX1 binding that promotes increased cytoplasmic free calcium levels and vasoconstriction,which may be an important mechanism of salt-sensitive hypertension.3.Chronic high salt diet could induce myocardial hypertrophy in salt-sensitive rats,and canagliflozin can significantly improve high salt-induced myocardial hypertrophy.4.SIRT3 is an important mechanism of high salt-induced myocardial hypertrophy and a therapeutic target of canagliflozin.