Cardioprotective effect of nicorandil, a mitochondrial ATP-sensitive potassium channel opener, prolongs survival in HSPB5 R120G transgenic mice

Abstract

Background: Transgenic (TG) mice with overexpression of an arg120gly (R120G) missense mutation in HSPB5 display desmin-related cardiomyopathy, which is characterized by formation of aggresomes. It is also known that progressive mitochondrial abnormalities and apoptotic cell death occur in the hearts of R120G TG mice. The role of mitochondrial dysfunction and apoptosis in disease progression, however, remains uncertain.

Methods and results: Mitochondrial abnormalities and apoptotic cell death induced by overexpression of HSPB5 R120G were analyzed in neonatal rat cardiomyocytes. Overexpression of mutant HSPB5 led to development of aggresomes with a concomitant reduction in cell viability in the myocytes. Overexpression of mutant HSPB5 induced a reduction in the cytochrome c level in the mitochondrial fraction and a corresponding increase in the cytoplasmic fraction in the myocytes. Down-regulation of BCL2 and up-regulation of BAX were detected in the myocytes expressing the mutant HSPB5. Concomitant with mitochondrial abnormality, the activation of caspase-3 and increased apoptotic cell death was observed. Cell viability was dose-dependently recovered in myocytes overexpressing HSPB5 R120G by treatment with nicorandil a mitochondrial ATP-sensitive potassium channel opener. Nicorandil treatment also inhibited the increase in BAX, the decrease in BCL2, activation of caspase-3 and apoptotic cell death by mutant HSPB5. To confirm the results of the in-vitro study, we analyzed the effect of nicorandil in HSPB5 R120G TG mice. Nicorandil treatment appeared to reduce mitochondrial impairment and apoptotic cell death and prolonged survival in HSPB5 R120G TG mice.

Conclusions: Nicorandil may prolong survival in HSPB5 R120G TG mice by protecting against mitochondrial impairments.

Figure 1. Effects of nicorandil (Nico) in neonatal rat cardiomyocytes expressing HSPB5 R120G (R120G).

(A) Typical pictures of Western blot analysis. Cardiomyocytes were infected with the adenovirus vector containing the wild-type HSPB5 (HSPB5) or R120G with or without Nico treatment. (B) Quantitative analysis of HSPB5 (n = 4). Values are the x-fold increase relative to cardiomyocyte cultures infected with the adenovirus vector containing LacZ whose values are arbitrarily set to 1. (C) Representative pictures of immunohistochemical analyses of the R120G-infected cardiomyocytes. HSPB5 (green) was detected by the anti-HSPB5 antibody as described in the Materials and methods section. To distinguish between the cardiomyocytes, cardiac troponin I was stained (red). (D) Typical picture of the filter assay for the detection of the aggregates. (E) Quantitative analysis of the aggregates containing the mutant R120G protein with or without Nico treatment (n = 4). (F) Representative pictures of immunohistochemical analyses in the R120G-infected cardiomyocytes. An amyloid oligomer (green) was detected by the anti-oligomer antibody as described in the Materials and methods section. (G) Quantitative analysis of the amyloid oligomer. Amyloid oligomer levels were measured by fluorescence intensity (n = 4). (H and I) Dot blotting shows the presence of the amyloid oligomer in the recombinant R120G protein with or without Nico treatment. (J) Protective effects of Nico on the cellular viability of the R120G infected cardiomyocytes. Cellular viability was determined by the MTT assay. Values are the x-fold increase relative to the cardiomyocyte infected with LacZ whose values are arbitrarily set to 1 (n = 6). *** p<0.001 vs. the cardiomyocytes infected with LacZ, # p<0.05 vs. cardiomyocytes infected with R120G.

Figure 2. Cytochrome c levels of mitochondrial fraction isolated from the cardiomyocytes expressing HSPB5 R120G (R120G) with or without Nico treatment.

(A) Typical pictures of Western blot analysis. Quantitative analysis of cytochrome c (B) and HSPB5 (C) in mitochondrial and cytosolic fractions isolated from the cardiomyocytes. GAPDH was used as a loading control in the cytosolic fraction and the voltage-dependent anion channel (VDAC) was used in mitochondrial fraction. *p<0.05, ** p<0.01, ***p<0.001 vs. the cardiomyocytes infected with LacZ (LacZ), ##p<0.01, ###p<0.001 vs. the cardiomyocytes infected with wild-type HSPB5 (HSPB5), ap<0.05 vs. cardiomyocytes infected with R120G treated with PBS. (n = 6).

Figure 3. Western blot analysis of apoptosis related proteins, BAX and BCL2, and apoptotic cell death in the cardiomyocytes expressing HSPB5 R120G (R120G) with or without Nico treatment.

(A) Typical pictures of Western blot analysis. (B) Quantitative analysis of BAX in mitochondrial fraction isolated from the cardiomyocytes. (C) Typical pictures of Western blot analysis. (D) Quantitative analysis of BCL2 in mitochondrial fraction isolated from the cardiomyocytes. (E) Caspase 3 activity in the cardiomyocytes. (F) Typical pictures of annexin V-positive cardiomyocytes. (G) Quantitative analysis of annexin V-positive cardiomyocytes. (H) Typical pictures of TUNEL-positive cardiomyocytes. (I) Quantitative analysis of TUNEL-positive cardiomyocytes. ** p<0.01, ***p<0.001 vs. the cardiomyocytes infected with LacZ (LacZ), ##p<0.01, ###p<0.001 vs. the cardiomyocytes infected with wild-type HSPB5 (HSPB5), ap<0.05, aaap<0.001 vs. cardiomyocytes infected with R120G treated with PBS. (n = 6).

Figure 4. Cell viability in cardiomyocytes expressing R120G.

(A) Protective effects of 10 µM Nico or 100 µM diazoxide (Diazo) on the cellular viability was inhibited by co-treatment with 500 µM 5-hydroxydecanoate (5HD). (B) No detectable effect on the cellular viability was seen by treatment with 10 µM sodium nitroprusside (SNP). Values are the x-fold increase relative to the cardiomyocyte infected with the LacZ whose values are arbitrarily set to 1. *** p<0.001, vs. the cardiomyocytes infected with LacZ (LacZ), ### p<0.001, vs. the cardiomyocytes infected with wild-type HSPB5 (HSPB5), aaa p<0.001 vs. cardiomyocytes infected with R120G treated with PBS, and bbb p<0.001 vs. cardiomyocytes infected with R120G treated with 10 µM Nico or 100 µM Diazo (n = 6).

Figure 5. Effect of Nico (81 mg/l of drinking water) on cardiac disease at a relatively early stage in the HSPB5 R120G (R120G) TG mice.

(A) Protocol was shown. Nico was administered via drinking water from 12 weeks for a total of 4 weeks in the R120G TG mice. (B) Typical pictures of Western blot analysis. (C) Quantitative analysis of HSPB5. Values are the x-fold increase relative to values in non-transgenic (NTG) mouse hearts whose values are arbitrarily set to 1. (D) Representative pictures of the immunohistochemistry are shown. (E) Typical picture of the filter assay for the detection of the aggregates containing HSPB5 proteins. (F) Quantitative analysis of the aggregates containing mutant R120G protein. (G) Fractional shortening assessed by the echocardiogram. Cardiac functional measurements were made at 16 weeks (n = 8 mice). (H) Typical pictures of Western blot analysis for ANF. (I) Quantitative analysis of ANF in the hearts in the R120G TG mice. (J) Masson's trichrome staining. (K) Cytochrome c levels of mitochondrial fraction isolated from the hearts from the R120G TG mice with or without Nico treatment. Typical pictures of Western blot analysis. Quantitative analysis of cytochrome c. GAPDH was used as a loading control in the cytosolic fraction and the voltage-dependent anion channel (VDAC) was used in the mitochondrial fraction. (L) Quantitative analysis of cytochrome c in mitochondrial and cytosolic fractions isolated from the hearts in the R120G TG mice. *p<0.05, ***p<0.001, vs. the NTG, #p<0.05 vs. the R120G TG mice (n = 6–8).

Figure 6. Effect of Nico (81 mg/l of drinking water) on cardiac disease at late stage in the HSPB5 R120G (R120G) TG mice.

(A) Protocol was shown. Nico was administered via drinking water from 20 weeks of age in the R120G TG mice. (B) Fractional shortening assessed by the echocardiogram. Cardiac functional measurements were made at 28 weeks (n = 6–10 mice). (C) The ratios of heart weight/body weight. (n = 6–10). (D) Representative pictures of the immunohistochemistry are shown. The aggregates containing the mutant R120G protein (upper panel green) and amyloid oligomer (lower panel green) were observed in the R120G TG mice. To distinguish between the cardiomyocytes, cardiac troponin I was stained (upper panel red) (E and F) Quantitative analysis of the aggregates containing the mutant HSPB5 R120G protein (E) and amyloid oligomer (F). (n = 4). (G) Masson's trichrome stain in the R120G TG mouse heart with or without Nico treatment. * p<0.05, *** p<0.001 vs. non-transgenic (NTG) mice; # p<0.05, ### p<0.001 vs. R120G TG mice.

Figure 7. Effect of Nico (81 mg/l of drinking water) on survival rate and biochemical parameters at the late stage in the HSPB5 R120G (R120G) TG mice.

(A) Survival curves. Nico effectively attenuated premature death in R120G TG mice. (B) Typical pictures of Western blot analysis for ANF. (C) Quantitative analysis of ANF in the hearts in the R120G TG mice. (D) Quantitative analysis of TUNEL-positive cardiomyocytes. (E) Typical pictures of Western blot analysis. (F) Quantitative analysis of HSPB5 and. Values are the x-fold increase relative to values in non-transgenic (NTG) mouse hearts whose values are arbitrarily set to 1. (D) Representative pictures of the immunohistochemistry are shown. The aggregates containing the mutant R120G protein are observed in R120G TG mice with or without Nico treatment. (E) Cytochrome c levels of mitochondrial fraction isolated from the R120G TG mouse heart with or without Nico treatment. (F) Typical pictures of Western blot analysis. Quantitative analysis of cytochrome c (F upper colomun) and HSPB5 (F lower column) in mitochondrial and cytosolic fraction isolated from the R120G TG mice. GAPDH was used as a loading control in the cytosolic fraction and voltage-dependent anion channel (VDAC) was used in the mitochondrial fraction. ***p<0.001 vs. non-transgenic (NTG) mice; # p<0.05, ### p<0.001 vs. R120G TG mice (survival rate: n = 19–20, and other experiments: n = 4).

Figure 8. Western blot analysis of apoptosis-related proteins, BAX and BCL2, and apoptotic cell death in HSPB5 R120G (R120G) TG mice with or without Nico treatment.

(A) Typical pictures of Western blot analysis. (B and C) Quantitative analysis of BAX (B) and BCL2 (C) in mitochondrial fraction isolated from the TG mouse hearts. * p<0.05, **p<0.01 vs. non-transgenic (NTG) mice; # p<0.05 vs. R120G TG mice (n = 4).