Selective inhibition of protein kinase C beta(2) attenuates inducible nitric oxide synthase-mediated cardiovascular abnormalities in streptozotocin-induced diabetic rats

Abstract

Objective: Impaired cardiovascular function in diabetes is partially attributed to pathological overexpression of inducible nitric oxide synthase (iNOS) in cardiovascular tissues. We examined whether the hyperglycemia-induced increased expression of iNOS is protein kinase C-beta(2) (PKCbeta(2)) dependent and whether selective inhibition of PKCbeta reduces iNOS expression and corrects abnormal hemodynamic function in streptozotocin (STZ)-induced diabetic rats.

Research design and methods: Cardiomyocytes and aortic vascular smooth muscle cells (VSMC) from nondiabetic rats were cultured in low (5.5 mmol/l) or high (25 mmol/l) glucose or mannitol (19.5 mmol/l mannitol + 5.5 mmol/l glucose) conditions in the presence of a selective PKCbeta inhibitor, LY333531 (20 nmol/l). Further, the in vivo effects of PKCbeta inhibition on iNOS-mediated cardiovascular abnormalities were tested in STZ-induced diabetic rats.

Results: Exposure of cardiomyocytes to high glucose activated PKCbeta(2) and increased iNOS expression that was prevented by LY333531. Similarly, treatment of VSMC with LY333531 prevented high glucose-induced activation of nuclear factor kappaB, extracellular signal-related kinase, and iNOS overexpression. Suppression of PKCbeta(2) expression by small interference RNA decreased high-glucose-induced nuclear factor kappaB and extracellular signal-related kinase activation and iNOS expression in VSMC. Administration of LY333531 (1 mg/kg/day) decreased iNOS expression and formation of peroxynitrite in the heart and superior mesenteric arteries and corrected the cardiovascular abnormalities in STZ-induced diabetic rats, an action that was also observed with a selective iNOS inhibitor, L-NIL.

Conclusions: Collectively, these results suggest that inhibition of PKCbeta(2) may be a useful approach for correcting abnormal hemodynamics in diabetes by preventing iNOS mediated nitrosative stress.

FIG. 1.

LY333531 treatment decreases PKCβ₂ phosphorylation and iNOS in cardiomyocytes. A: (top) Representative Western blot showing phospho-PKCβ₂ (Thr⁶⁴¹) in comparison with total PKCβ₂ in cardiomyocytes exposed to low glucose (LG; 5.5 mmol/l) or high glucose (HG; 25 mmol/l) for 18 h in the absence or presence of LY333531 (LY333531; 20 nmol/l). (bottom) The densitometric values of phospho-PKCβ₂ were normalized to corresponding total PKCβ₂ densitometric values and the relative band intensities are expressed as means ± SE (n = 4 independent experiments). *P < 0.05 compared with low glucose, low glucose + LY333531, and high glucose + LY333531 groups. B: Representative Western blot showing iNOS expression, with glyceraldehyde-3-phosphate dehydrogenase (GADPH) as a loading control, in cardiomyocytes exposed to low (5.5 mmol/l) or high glucose (25 mmol/l) for 18 h in the presence or absence of LY333531 (20 nmol/l). iNOS densitometric values were normalized to their corresponding GAPDH densitometric values and expressed as relative band intensities. All data are expressed as means ± SE (n = 3 to 5 independent experiments). *P < 0.05 compared with low glucose, low glucose + LY333531, and high glucose + LY333531 groups.

FIG. 2.

Effect of LY333531 (LY)on PKCβ₂ phosphorylation and iNOS expression in control and diabetic heart tissues. A: Representative Western blot showing phospho-PKCβ₂ (Thr⁶⁴¹) in comparison with total PKCβ₂ expression levels in ventricular tissues of control (C), diabetic (D), control LY333531-treated (C-LY), and diabetic LY333531-treated (D-LY) rats. The densitometric values of phospho-PKCβ₂ were normalized to corresponding total PKCβ₂ expression levels and the relative band intensities are expressed as means ± SE (n = 5). *P < 0.05 compared with C, C-LY, and D-LY groups. B: Representative Western blot showing iNOS expression in the ventricular tissues of C, D, C-LY, and D-LY groups. iNOS protein was immunoprecipitated from heart ventricular tissue lysates using mouse monoclonal anti-iNOS antibody. Equal amounts of immunoprecipitated complex were loaded on to the gels and were subjected to SDS-PAGE. Densitometric data are expressed as percentage of control. All values are expressed as means ± SE (n = 4). *P < 0.05 compared with C, C-LY, and D-LY groups.

FIG. 3.

Effect of LY333531 (LY) on PKCβ activation and iNOS expression in isolated superior mesenteric arteries and cultured rat aortic VSMC. A: Representative Western blot showing iNOS expression in superior mesenteric arteries of control (C), diabetic (D), control LY333531-treated (C-LY), and diabetic LY333531-treated (D-LY) groups. iNOS was immunoprecipitated from superior mesenteric artery tissue lysates using mouse monoclonal anti-iNOS antibody. Densitometric data are expressed as percentage of control. All values are expressed as means ± SE (n = 4). *P < 0.05 compared with control (C), control LY333531-treated (C-LY), and diabetic LY333531-treated (D-LY) groups; ^#P < 0.05 compared with control and control LY333531-treated. B: Representative Western blot showing iNOS expression, with GAPDH shown as a loading control, in aortic VSMC exposed to low glucose (LG; 5.5 mmol/l) and high glucose (HG; 25 mmol/l) for 36 h in the presence or absence of LY333531 (20 nmol/l). iNOS densitometric values were normalized to their corresponding GAPDH densitometric values and expressed as relative band intensities. All data are expressed as means ± SE (n = 4 independent experiments). *P < 0.05 compared with low glucose, low glucose + LY333531, and high glucose + LY333531 groups. C: Representative Western blot showing phospho- and total PKCβ₂ expression in VSMC exposed to low (5.5 mmol/l) or high glucose (25 mmol/l) for 36 h in the presence or absence of LY333531 (20 nmol/l). The densitometric values of phospho-PKCβ₂ were normalized to corresponding total PKCβ₂ expressionlevels and the relative band intensities are expressed as means ± SE (n = 4 independent experiments). *P < 0.05 compared with low glucose, low glucose + LY333531, and high glucose + LY333531 groups.

FIG. 4.

Effect of PKCβ₂ suppression on PKCβ₂ expression and activation and iNOS expression in VSMC. Cells were transfected with either MOCK or PKCβ₂-specific siRNA and exposed to low or high glucose for 36 h. A: Representative Western blot showing total and phospho-PKCβ₂ expression, with GAPDH as a loading control in VSMC treated with MOCK or PKCβ₂ siRNA. The densitometric values of PKCβ₂ were normalized to corresponding total GAPDH densitometric values and the relative band intensities are expressed as means ± SE (n = 4). Open bars represent low glucose–treated and dark bars represent high glucose–treated VSMC. *P < 0.05 compared with MOCK siRNA–treated VSMC exposed to low glucose or high glucose conditions, @P < 0.05 compared with all other groups. B: Representative Western blot showing iNOS expression, with GAPDH as a loading control in VSMC treated with MOCK or PKCβ₂ siRNA. The densitometric values of iNOS were normalized to corresponding total GAPDH densitometric values, and the relative band intensities are expressed as means ± SE (n = 4). Open bars represent low glucose–treated and dark bars represent high glucose–treated VSMC. *P < 0.05 compared with all other groups.

FIG. 5.

Effect of LY333531 (LY) on ERK1/2 and NF-κB activation in VSMC. A: Representative Western blot showing phospho-ERK-1 (Thr²⁰²) and phospho-ERK-2 (Tyr²⁰⁴) in comparison with total ERK1/2 and with GAPDH as a loading control in VSMC exposed to low glucose (LG; 5.5 mmol/l) or high glucose (HG; 25 mmol/l) for 36 h in the presence or absence of LY333531 (20 nmol/l). The densitometric values of phospho ERK-1 were normalized to corresponding total ERK-1densitometric values and the relative band intensities are expressed as means ± SE (n = 4 independent experiments). *P < 0.05 compared with all other groups. B: Representative Western blot showing phospho-NF-κB P65 subunit (Ser⁵³⁶) in comparison with total NF-κB P65 and with GAPDH as a loading control in VSMC exposed to low (5.5 mmol/l) and high (25 mmol/l) glucose for 36 h in the presence or absence of LY333531 (20 nmol/l). The densitometric values of phospho-NF-κB were normalized to corresponding total phospho-NF-κB P65densitometric values and the relative band intensities are expressed as means ± SE (n = 4 independent experiments). *P < 0.05 compared with all other groups.

FIG. 6.

Effect of PKCβ₂ silencing on ERK1/2 and NF-κB activation in VSMC. A: Representative Western blot showing phospho-ERK-1 (Thr²⁰²) and phospho-ERK-2 (Tyr²⁰⁴) in comparison with total ERK1/2 in VSMC treated with MOCK or PKCβ₂ siRNA. The densitometric values of phospho-ERK-1 were normalized to corresponding total ERK-1 densitometric values, and the relative band intensities are expressed as means ± SE (n = 4 independent experiments). Open bars represent low glucose–treated and dark bars represent high glucose–treated VSMC. *P < 0.05 compared with all other groups. B: Representative Western blot showing phospho-NF-κB P65 subunit (Ser⁵³⁶) in comparison with total NF-κB P65 in VSMC treated with MOCK or PKCβ₂ siRNA. The densitometric values of phospho-NF-κB P65 were normalized to corresponding total NF-κB P65 densitometric values and the relative band intensities are expressed as means ± SE (n = 4 independent experiments). Open bars represent low glucose–treated and dark bars represent high glucose–treated VSMC. *P < 0.05 compared with all other groups.

FIG. 7.

Effect of 3 weeks of LY333531 (LY) (1 mg/kg/day) or L-NIL (3 mg/kg/day) treatment on hemodynamic parameters in freely moving conscious rats. Data represent means ± SE (n = 8). A: Effect of LY333531 or L-NIL on MABP. *P < 0.05 compared with all other groups. B: Effect of LY333531 or L-NIL on heart rate. *P < 0.05 compared with all other groups. C: Effect of LY333531 or L-NIL on endothelial function as measured by the change in blood pressure (ΔMABP) in response to a single intravenous bolus dose of l-NAME (10 mg/kg) in control (C) and diabetic (D) rats. *P < 0.05 compared with their respective control groups. #P < 0.05 compared with diabetic and diabetic + LY333531 groups. D: Effect of LY333531 or L-NIL on pressor responses to bolus doses of methoxamine (100–300 nmoles/kg). *P < 0.05 compared with all other groups; #P < 0.05 compared with diabetic rats.