Acute hyperglycemia suppresses left ventricular diastolic function and inhibits autophagic flux in mice under prohypertrophic stimulation

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: Acute hyperglycemia suppresses diastolic function, damages mitochondrial energy signaling, and inhibits autophagic flux in prohypertrophic factor-stimulated cardiomyocytes.

Keywords: Acute hyperglycemia; Autophagic flux; Diastolic function; Myocardial hypertrophy.

Fig. 1

Diastolic dysfunction appears in the early phase of pressure overload. After 30 min of transverse aortic constriction (TAC) in C57 mice, invasive hemodynamics were evaluated. a Left ventricular systolic pressure (LVSP). b Left ventricular end-diastolic pressure (LVEDP). c Heart rate. d Maximum rate of change in left ventricular pressure (dp/dt max). e Minimum rate of change in left ventricular pressure (dp/dt min). f Exponential time constant of relaxation (τ). g dp/dt max corrected with LVSP. h dp/dt min corrected with LVSP. *P < 0.05 versus the sham group, n = 6 and 9 in the sham and TAC groups, respectively

Fig. 2

Diastolic dysfunction and insulin resistance appear in mice with cardiac hypertrophy. After 2 weeks of TAC in C57 mice, invasive hemodynamics were evaluated. a Representative recordings of left ventricular pressure and the rate of change in pressure. b Left ventricular systolic pressure (LVSP). c Left ventricular end-diastolic pressure (LVEDP). d Maximum rate of change in left ventricular pressure (dp/dt max) corrected by LVSP. e Minimum rate of change in left ventricular pressure (dp/dt min) corrected by LVSP. f Heart to body weight ratio (HW/BW) and lung to body weight ratio (LW/BW). *P < 0.05 versus the sham group, n = 9 in each group

Fig. 3

Insulin resistance appears in mice with TAC for 2 weeks. a Plasma glucose concentrations in response to an intraperitoneal glucose tolerance test (IGTT, after fasting for 14 h, glucose 2 g/kg, ip). The insert shows fasting glucose levels. b Time course of plasma glucose concentrations after insulin injection. c Linear correlation between HOMA-IR and heart weight to body weight ratio (HW/BW) in the TAC and sham mice groups. *P < 0.05 versus the corresponding sham group, n = 16 and 12 in the sham and TAC groups, respectively, (panels a, b); n = 24 for panel c

Fig. 4

Acute hyperglycemic stress suppresses diastolic function in mice with TAC for 2 weeks. a Representative time course recordings of left ventricular pressure (LVP) and the rate of rise and fall rate of LV (dp/dt). b Time course of left ventricular systolic pressure (LVSP). c Time course of left ventricular end-diastolic pressure (LVEDP). d Time course of corrected dp/dt max in response to glucose overload (2 g/kg, ip). e Time course of corrected dp/dt min in response to glucose overload. *P < 0.05 vs. the corresponding time point in the sham group, n = 5 per group. f Western blot analysis of LC3B-II and P62. *P < 0.05 vs. sham group; ^# P < 0.05 vs. TAC group, n = 5 per group

Fig. 5

High glucose levels downregulate PGC-1α and inhibit autophagic flux. a Representative pictures of cultured neonatal rat cardiomyocytes (NRCs) stained with β-actin plus DAPI staining of the nucleus in response to angiotensin II stimulation with/without high glucose stimulation. Western blot analysis of PGC-1α (b, c), LC3B-II (d, e), and P62 (d, f) expression in cultured NRCs. *P < 0.05 vs. control group, ^# P < 0.05 vs. angiotensin II group. Each experiment was repeated four times

Fig. 6

High glucose levels inhibit autophagic flux in cultured neonatal rat cardiomyocytes. a Western blot images of LC3B-II and P62 in cultured neonatal rat cardiomyocytes (NRCs) in the presence of bafilomycin A1. b Semi-quantitation of LC3B-II and P62 expression levels. c Representative immunofluorescent NRCs expressing mRFP-GFP-LC3. d Semi-quantitation of autophagosomes (yellow) and autolysosomes (red). *P < 0.05 vs. the autophagosomes in the corresponding group without bafilomycin A1 treatment; ^# P < 0.05 vs. the autolysosomes in the corresponding group without bafilomycin A1 treatment. Experiments were repeated four times. G5.5, glucose 5.5 mmol/L; G25, glucose 25 mmol/L; Ang II, angiotensin II; Baf, bafilomycin A1

Fig. 7

Chronic hyperglycemia levels induce LV diastolic dysfunction and inhibit autophagic flux in mice at 6 weeks after induction of type 1 DM. a Plasma glucose concentrations. b Representative images of H&E (top) and Masson stained (bottom) myocardial tissue. Scale bar = 10 µm. c Left ventricular end-diastolic pressure (LVEDP). d Maximum of change in left ventricular pressure (dp/dt max). e Minimum rate of change in left ventricular pressure (dp/dt min). f Exponential time constant of relaxation (τ). g, h Western blot analysis of PGC-1α, LC3B-II, and P62 expression in myocardial tissues. *P < 0.05 vs. the sham group, n = 5 per group

Fig. 8

Proposed hyperglycemia-mediated diastolic dysfunction signaling pathway. The solid and dotted arrows indicate evidence from our study and that from previous literature, respectively. ROS reactive oxygen species; uparrow activation and; inhibition