Acute hyperglycemia suppresses left ventricular diastolic function and inhibits autophagic flux in mice under prohypertrophic stimulation
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Left ventricular (LV) dysfunction is closely associated with LV hypertrophy or diabetes, as well as insufficient autophagic flux. Acute or chronic hyperglycemia is a prognostic factor for patients with myocardial infarction. However, the effect of acute hyperglycemia on LV dysfunction of the hypertrophic heart and the mechanisms involved are still unclear. This study aimed to confirm our hypothesis that either acute or chronic hyperglycemia suppresses LV diastolic function and autophagic flux.
The transverse aortic constriction (TAC) model and streptozocin-induced type 1 diabetic mellitus mice were used. LV function was evaluated with a Millar catheter. Autophagic levels and autophagic flux in the whole heart and cultured neonatal rat cardiomyocytes in response to hyperglycemia were examined by using western blotting of LC3B-II and P62. We also examined the effect of an autophagic inhibitor on LC3B-II and P62 protein expression and LC3 puncta.
In mice with TAC, we detected diastolic dysfunction as early as 30 min after TAC. This dysfunction was indicated by a greater LV end-diastolic pressure and the exponential time constant of LV relaxation, as well as a smaller maximum descending rate of LV pressure in comparison with sham group. Similar results were also obtained in mice with TAC for 2 weeks, in addition to increased insulin resistance. Acute hyperglycemic stress suppressed diastolic function in mice with myocardial hypertrophy, as evaluated by invasive LV hemodynamic monitoring. Mice with chronic hyperglycemia induced by streptozocin showed myocardial fibrosis and diastolic dysfunction. In high glucose-treated cardiomyocytes and streptozocin-treated mice, peroxisome proliferator-activated receptor-γ coactivator 1α was downregulated, while P62 was upregulated. Autophagic flux was also significantly inhibited in response to high glucose exposure in angiotensin-II treated cardiomyocytes.
Acute hyperglycemia suppresses diastolic function, damages mitochondrial energy signaling, and inhibits autophagic flux in prohypertrophic factor-stimulated cardiomyocytes.
KeywordsAcute hyperglycemia Diastolic function Myocardial hypertrophy Autophagic flux
transverse aortic constriction
peroxisome proliferator-activated receptor-γ coactivator 1α
light chain 3 beta-II
ubiquitin-binding autophagy receptor
LV systolic pressure
LV end diastolic pressure
homeostasis model assessment for insulin resistance
Left ventricular (LV) diastolic dysfunction is present in various cardiovascular diseases such as hypertension , myocardial hypertrophy [2, 3, 4], coronary heart disease , and diabetes mellitus (DM) [6, 7]. Worsening of diastolic dysfunction is a predictor of mortality in cardiovascular diseases and some severe non-cardiovascular diseases, including septic shock and acute pancreatitis [8, 9, 10]. The detrimental role of chronic hyperglycemia and DM on diastolic function has been well recognized . The prognostic value of acute hyperglycemia in patients with acute myocardial infarction has also attracted attention of clinical investigators . However, whether acute hyperglycemia is a factor that worsens diastolic dysfunction is unclear, and if so, the underlying mechanisms are unknown. Addressing these issues is of clinical importance for avoiding deterioration of diastolic dysfunction in patients with severe diseases.
Glucose deprivation causes autophagy. Therefore, hyperglycemia is likely to inhibit autophagy . Insufficient autophagy is thought to cause or promote heart failure [14, 15, 16]. However, a previous report showed that increased autophagy contributes to LV diastolic dysfunction in pulmonary arterial hypertension . Glucose is the main source of energy in the failing heart. Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, a transcriptional coactivator, is beneficial for heart failure by inducing a change in phenotype towards oxidative metabolism of energy . The association between PGC-1α and autophagy in cardiomyocytes that are exposed to high glucose is unclear. However, several lines of evidence in non-cardiomyocytes have shown that PGC-1α promotes autophagy [19, 20]. Based on all of these findings, we hypothesize that acute hyperglycemic stress suppresses diastolic function in preexistent heart disease by downregulating PGC-1α and inhibiting autophagic flux.
In the present study, we investigated the effect of acute hyperglycemia on LV diastolic function in a pressure-overload model with preexistent diastolic dysfunction and insulin resistance. We also investigated the potential mechanisms related to PGC-1α and autophagic flux in cultured cardiomyocytes. Although echocardiographic evaluation of LV diastolic dysfunction is a routine procedure in clinical practice, its accuracy is inferior to cardiac catheterization. Therefore, we used invasive LV hemodynamic parameters to evaluate diastolic function in this study.
C57BL/6 male mice (9–10 weeks old) were subjected to TAC or sham operation as previously described [2, 21]. Briefly, prior to surgery, mice were anesthetized with a combination of ketamine (100 mg/kg) and xylazine (5 mg/kg) via intraperitoneal injection (ip), after intubation and artificial respiration, the chest-open was then opened via the second intercostal space to access the aorta. A non-absorbable 7–0 silk suture was tied around the aorta between the right innominate and left carotid arteries. This caused a constriction of approximately 65 % using a 27 gauge-needle as a guide. For sham operations to serve as controls, mice received the same surgical procedure, except that the suture around the aorta was removed prior to closing the chest cavity. At various time points (1, 2 and 4 weeks) mice were sacrificed with an overdose of pentobarbital (150 mg/kg). The hearts and lungs were dissected to measure organ weight and to perform histological analysis.
Type 1 DM model
Ten-week-old male C57BL/6 mice were purchased and injected intraperitoneally with streptozocin (STZ) (Sigma Chemicals, St. Louis, USA) 70 mg/kg/day for 5 days. STZ was dissolved in 10 mmol/L sodium citrate buffer (pH 4.5). Control mice were injected with the buffer alone. Three days later, mice with a random blood glucose level >20 mmol/L were assigned to the hyperglycemic groups, whereas citrate buffer-treated mice were assigned to the control group. Six weeks later, mice were sacrificed with an overdose of pentobarbital (150 mg/kg), and the hearts were harvested for histological and molecular examinations.
Invasive LV hemodynamic measurements
Thirty minutes or 2 weeks after surgery, mice were anesthetized, intubated, and ventilated as mentioned above. A 1.4F Millar catheter (Millar Instruments, Inc., Houston, TX) was then inserted into the right carotid artery and advanced into the LV cavity. LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP), maximum and minimum rates of change in LV pressure (dp/dt max and dp/dt min, respectively) were recorded. The exponential time constant of LV relaxation (τ) was calculated using Power Lab software (blood pressure module; AD Instruments, Shanghai Trading Co, Shanghai, China). Some mice were treated with high glucose or mannitol (2 g/kg ip) and the time-course of LV hemodynamics was recorded. For mice that received a high glucose load test, overnight fasting was performed before the test to avoid xylazine/ketamine anesthesia-induced hyperglycemia .
Blood glucose measurements
Plasma glucose (fasting or in response to high glucose or insulin treatment) measurements were performed in all mice using a standard glucometer (Accu-Chek, Roche, Mannheim, Germany). Whole blood samples (3 μl) were taken from mouse tails with a glucose sensor inserted in the glucometer. Plasma glucose concentrations were read 30 s later. Serum insulin levels were measured according to the protocol provided by the manufacturer (EIA-3440 enzyme linked immunosorbent assay kit; Diagnosis-related Group, Germany). Homeostasis model assessment for insulin resistance (HOMA-IR) values were determined from results of the fasting blood glucose (FBG) and fasting insulin (FINS) tests, using the equation HOMA-IR = [FBG (mg/dl, 1 mmol/L = 18 mg/dL) × FINS (ng/ml)]/22.5.
Ventricular myocytes were prepared from Sprague–Dawley rats (age, 1–2 days), which were obtained from the Animal Center of Southern Medical University. In brief, the rats were sacrificed by 2 % isoflurane inhalation. The hearts were quickly excised and immediately embedded in freezing Hank’s solution. Cardiomyocytes were dispersed by digestion with 0.1 % trypsin and 0.03 % collagenase at 37 °C. The cells were then collected after differential adhesion of non-cardiomyocytes and plated at a density of 150–200 cells/mm2. Cardiomyocytes were incubated for 48 h in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal calf serum and then grown for 24 h under serum free conditions. Cells were treated with angiotensin II (1 μmol/L) and/or high-glucose solution (25/50 mmol/L), and collected for protein extraction at 24 h.
Protein was obtained from cultured cardiomyocytes or hearts from STZ-treated mice, and was extracted in radio-immunoprecipitation assay lysis buffer. Samples were loaded onto 10 % sodium dodecyl sulfate–polyacrylamide gels and the protein was transferred to polyvinyl difluoride membranes. Immunoblotting was then performed by using PGC-1α antibody (ab191838; Abcam, Shanghai, China) or autophagy antibodies (light chain 3 beta [LC3B]-I/II, 2775s; and P62, 5114s; Cell Signaling Technology, Danvers, MA), and GAPDH antibody (ARG10112; Arigo, Taiwan, China). Immunoreactive bands were visualized by the enhanced chemiluminescence method (Amersham, Piscataway, NJ) with a western blotting detection system (Kodak Digital Science, Rochester, NY). These bands were quantified by densitometry with Scion Image software (Image J 1.42q; NIH, Bethesda, MD). We used the LC3B-II/loading control ratio rather than the LC3B II/LC3B-I ratio for qualification of LC3-II expression levels according to a newly published guideline .
Assay of autophagic flux
To measure autophagic flux, cardiomyocytes were treated with bafilomycin A1 (Selleck; Texas, USA), a lysosomal inhibitor, at 100 nmol/L for 24 h. Cellular autophagic flux was estimated by western blots of LC3B-II and P62 protein as well as by tandem fluorescent mRFP-GFP-LC3. Cultured cardiomyocytes were infected with lentivirus carrying mRFP-GFP-LC3 (Cat. No. GPL2001; Genechem Co., Shanghai, China) for 72 h (multiple of infection = 25). subsequently, cells were either treated with angiotensin II (1 μmol/L), glucose (5.5 or 25 μmol/L), or bafilomycin A1 (100 nmol/L) for 24 h. Cells were then washed with PBS, fixed with 4 % paraformaldehyde and viewed under confocal laser scanning microscopy (Olympus FV1000; Japan). In merged images, puncta in autophagosome appeared yellow, while puncta in autolysosomes appeared red.
All data analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL). Data are presented as mean ± standard error of the mean. Comparisons were made using unpaired Student’s t tests and one-way or two-way ANOVA, as appropriate. P-values less than 0.05 were considered statistically significant.
Diastolic dysfunction appears in the early phase of pressure overload
Diastolic dysfunction and insulin resistance appear in mice with cardiac hypertrophy
Acute hyperglycemic stress suppresses diastolic function in mice with preexistent diastolic dysfunction
High glucose levels downregulate PGC-1α and inhibit autophagic flux
Chronic hyperglycemia induces LV diastolic dysfunction and inhibits autophagic flux
Acute hyperglycemia is a transient hyperglycemia referred to as stress-induced hyperglycemia. Acute hyperglycemia is commonly observed on admission and during hospitalization for cardiovascular diseases, and non-cardiovascular diseases, such as traumatic injury, burns and surgical intervention. This condition is associated with an increase in morbidity and mortality compared with hospitalized patients with normal glucose levels . A recent report by Baranyai et al.  demonstrated that cardioprotection exerted by remote ischemic preconditioning can be abolished by acute hyperglycemia. Mebazaa et al.  reported that hyperglycemia is a prognostic predictor for mortality of patients with acute heart failure. They found a 9 % increase in the risk of 30-day mortality for every 1 mmol/L increase in blood glucose concentration in patients with acute heart failure.
With regard to the effect of chronic hyperglycemia on the heart, Rubin et al.  reported that chronic hyperglycemia contributes to subclinical myocardial damage in persons without clinically evident coronary heart disease. This clinical finding is in agreement with our results that STZ-induced chronic hyperglycemia suppressed LV diastolic function in mice without preexistent cardiovascular disease.
Pre-clinical diastolic dysfunction is prevalent and will progress to symptomatic heart failure . In patients with DM or hypertension, diastolic dysfunction is a risk of progression to heart failure and death [32, 33]. In agreement with previous studies [3, 34], in our study, we found that fasting glucose and glucose tolerance were impaired in hypertensive mice induced by TAC. This situation may be one of the reasons for diastolic dysfunction. Catena et al.  reported that impaired fasting glucose and glucose tolerance were associated with more prominent diastolic impairment in uncomplicated hypertensive patients, which is in consist with findings in our study.
Mitochondrial dysfunction of cardiomyocytes has been implicated in heart failure of diverse etiologies. PGC-1α is a master regulator of mitochondrial biogenesis and breathing, and downregulation or loss of PGC-1α is detrimental for heart failure [36, 37]. Hyperglycemia-induced reactive oxygen species in mitochondria play a critical role in the development of complications from diabetes. Overexpression of PGC-1α completely blocks hyperglycemia-induced production of mitochondrial reactive oxygen species and promotion of mitochondrial biogenesis . Based on these lines of evidence, our study suggests high glucose-downregulated PGC-1α contributes to diastolic dysfunction. Recent studies have shown that stimulating mitochondrial function or control of glucose with voglibose or sitagliptin improves diastolic dysfunction [39, 40].
Diastolic dysfunction exists in the hypertrophied heart. Autophagy is diminished in response to pressure overload or β-adrenergic stimulation, although protein turnover is increased during hypertrophy [41, 42]. Wang et al.  reported that low concentrations of angiotensin II induce autophagy while high concentrations diminish autophagy in cultured cardiomyocytes, which is in agreement with our results. Appropriated levels of autophagic flux are thought to maintain cellular homeostasis and cell survival, which are beneficial for heart failure [44, 45]. Numerous studies have demonstrated that autophagy is impaired in the heart under DM , which is also in agreement with our findings. Zhang et al.  recently reported that an increase in autophagy prevents cardiac fibrosis and inflammation in type 1 DM mice. Another study showed that caloric restriction improves diastolic dysfunction in diabetic rat hearts by enhancing autophagy . In our study, we observed that acute stimulation with high glucose levels rapidly downregulated LC3B-II and upregulated P62. This finding suggested inhibition of an autophagy by reducing formation of autophagosomes. Zhang et al.  reported that induction of autophagosome can be as fast as 15 min after treatment of retinal pigment epithelial cells with the drug N-retinyl-N-retinylidene ethanolamine.
In contrast to acute hyperglycemia, chronic hyperglycemia increased myocardial LC3B-II level. This situation could be due to long-term inhibition of autophagic flux as shown by upregulation of P62. Kanamori et al.  reported diastolic impairment and in increase of LC3 II and P62 in mice with type 1 DM, which is consistent with our findings. However, the differences between acute and chronic hyperglycemia on autophagic activity need to be further clarified. Taken together, our findings suggest that reactivation of autophagy is likely to improve diastolic dysfunction induced by myocardial hypertrophy and DM.
Acute hyperglycemia suppresses diastolic function, damages mitochondrial energy signaling, and inhibits autophagic flux in prohypertrophic factor-stimulated cardiomyocytes. Our findings suggest that preventing acute hyperglycemia is of clinical importance for avoiding deterioration of diastolic dysfunction in patients with LV hypertrophy. Modulation of autophagy may be a novel strategy for improving diastolic dysfunction induced by myocardial hypertrophy and DM.
All of the authors have made an important contribution to the study and are thoroughly familiar with the original data. The contribution of each author is as follows: (1) conception and design (YL, KC, MK), (2) conducting the experiment (YL, JX, HH, YZ, KC) (3) analysis and interpretation of data (YL, XH, SC, WL, JB, JX, HH, YZ, ZC, HL, KC, MK), (4) drafting of the manuscript or revising it critically for important intellectual content (YL, KC, YZ); (5) All authors read and approved the final manuscript.
This study was supported by Grants from the National Natural Science Foundation of China (No. 81170146, No.81570464), and the Provincial Natural Science Foundation of Guangdong (2014A030313342, 2015A030313301, 2015A030313298).
Competing financial interests
The authors declare that they have no competing financial interests.
Availability of data and material
Consent for publication
If the manuscript is accepted, we approve of it for publication in Cardiovascular Diabetology.
Ethics approval and consent to participate
Approval for this study was granted by our university ethics review board. All procedures were performed in accordance with our Institutional Guidelines for Animal Research (Nanfang Hospital, Southern Medical University) and complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).
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