| Objective:Chronic high dietary salt intake is closely associated with elevated blood pressure level and target organ injuries. In our previous work, we use8.0%NaCl loaded spontaneously hypertensive rats (SHR) and find that8-12weeks is the key time window in which a transition from compensated to decompensated left ventricular (LV) hypertrophy occur. In addition, lots of vacuole-like structures were observed in12w SHR and there are more vacuoles in high salt (HS)-loaded SHR compared with their low salt (LS)-fed counterparts, indicating a high salt intake-related phenomenon. Therefore, the present work was designed to demonstrate the mechanism of vacuole-like structures formation and to investigate their association with high salt intake induced LV dysfunction.Methods:Seven-week-old male SHR and Wistar Kyoto (WKY) rats were randomized fed LS (0.5%NaCl) and HS (8.0%NaCl) diets. At the end of8,12,16weeks, invasive hemodynamic measurements were performed. After perfusing with precooled physiological saline the hearts were quickly removed. Samples from left ventricular (0.5×0.5×1cm3) were fixed in2.5%glutaraldehyde for evaluate the ultra-structure by transmission electron microscope. Other samples were tored in-80℃for further study. After embeded and sectioned, cross sections were stained in a routine fashion with hematoxylin and eosin (HE), oil red O, alcian blue/periodic acid-schiff (AB-PAS), toluidine blue. Interstitial collagen deposition was estimated with trichrome Masson staining. Mean cardiomyocyte cross-sectional area was determined with wheat germ agglutinin (WGA). A global activation of autophagy-associated key components and myocardial tonicity-responsive enhancer binding protein (TonEBP) were detected by western blot. In vitro study, H9c2cardiomyocytes were treated with NaCl (final action concentrations were25mM,50mM,100mM,150mM, equal to350mOsm/L,400mOsm/L,500mOsm/L,600mOsm/L) in complete growth medium for24hours, plus CoroNa Green then detect fluorescence signal with flow cytometry. It is reported that high salt loading with a diet containing8.0%NaCl in Sprague-Dawley rats for2weeks led to an increase of [Na+]-40mM in the skin. Based on our NaCl concentration gradient study, we used an additional50mM NaCl (equal to400mOsm/L) in the culture medium to mimic the extracellular hyperosmolarity induced by chronic high salt intake in the subsequent in vitro studies. H9c2cardiomyocytes were treated with normal and50mM NaCl medium, a global activation of autophagy-associated key components were detect by PCR and Western blot. In addition, apoptosis were detected with Annexin V-FITC. H9c2cardiomyocytes were treated with hyperosmotic medium including50mM NaCl,50mM CsCl, or100mM mannitol (400mOsm/L), ROS scavenger N-Acety-L-cysteine (NAC)(0.5mM,2.0mM), and then the level of ROS generation and the level of autophagy were studied using flow cytometry, real-time quantitative PCR and western blot analysis.Results:Throughout the16weeks of dietary intervention, there was a significant decrease in body weight in the SHR fed with the HS diet. At12weeks and16weeks of dietary intervention, the HS diet led to an increase in left ventricular mass, cardiomyocyte hypertrophy and collagen deposition in SHR and WKY compared with their low salt-fed counterparts. Serial invasive LV hemodynamic analysis revealed that a HS diet progressively impaired the LV systolic (SBP and+dP/dtmax) and diastolic (LVEDP and-dP/dtmin) functions of the SHR in a time-dependent manner. There results demonstrated that a transition from compensated LV hypertrophy to decompensation occurred when the HS diet intervention persisted for12weeks in the SHR. A time-dependent increase in collagen deposition and LVEDP was also noted in the LS-fed SHR and HS-fed WKY rats, but an obvious deterioration of the LV systolic and diastolic functions was not observed during this period. Using various staining methods and TEM for pathological examinations, we demonstrated that the cardiac vacuoles observed in the HS-fed SHR under light microscopy are the results of autophagic vacuolization. In addition, serial western blot analysis of the heart tissue revealed a global activation of the autophagy-related proteins (Beclinl, Atg5-Atg12, LAMP1, LC3II) in the HS-fed SHR that were sacrificed after12weeks of salt loading. This activation persisted to16weeks in SHR on the HS diet, which demonstrated a temporal and spatial correlation between autophagy activation and LV function deterioration in the HS-fed SHR. Similarly, autophagy activation was observed in the HS-fed WKY. Western blot evidence of increased myocardial TonEBP in the HS-fed WKY and SHR. In vitro experiment, we found that there was a proportional increase of cytosolic [Na+] as extracellular [Na+] increased. In addition, cubation with an additional50mM NaCl induced an enhanced ROS production, pro-apoptotic response and the upregulation of key components of autophagy at both the mRNA and protein levels. When50mM NaCl was replaced by50mM CsCl in the culture medium, the ROS production was significantly reduced, implying that the increased extracellular NaCl induced enhancement of H9c2ROS generation is primarily dependent on Na+. When NAC was used as an ROS scavenger, the high NaCl induced upregulation of key autophagy components was significantly attenuated, implying that high [Na+]-induced autophagy activation is ROS dependent. Interestingly, when100mM mannitol was used as a positive control at the same extracellular hyperosmolality with50mM NaCl, the magnitude of ROS generation and the pattern of key autophagic component upregulation were different from those achieved by50mM NaCl, providing further evidence to indicate that high [Na+]-induced changes in H9c2cells are not entirely dependent on extracellular osmolality.Conclusion:This work depicted the kinetics of myocardial autophagy during the LV aggravation after HS challenge in SHR and revealed a novel mechanism by which interstitial hypertonicity-induced cytosolic [Na+] elevation triggers ROS-dependent autophagy activation. |
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