Adenosine regulation of microtubule dynamics in cardiac hypertrophy

Abstract

There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics.

Fig. 1.

2-Chloroadenosine (CADO) or adenosine (ADO) reduces cytoskeletal tubulin. A: neonatal rat ventricular myocytes were treated with 50 μM phenylephrine (PE) in the presence or absence of 5 μM 2-chloroadenosine for 48 h. Cells were split into Triton-soluble (Sol) and insoluble [cytoskeletal (CSK)] fractions, separated on 9% polyacrylamide gel, and probed for α-sarcomeric actin, desmin, α-tubulin, and GAPDH. B: adenosine reduces cytoskeletal tubulin. Cells were treated with 50 μM PE + 10 μM adenosine + 1 μM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) for 48 h. C: scanning densitometry was used to quantitate the level of expression of each protein in the different fractions (n = 8 for sarcomeric actin, n = 8 for β-actin, n = 2 for desmin, n = 12 for tubulin). †P < 0.05 vs. PE. *P ≤ 0.05 vs. control; **P < 0.01 vs. control; ††P < 0.01 vs. PE.

Fig. 2.

2-Chloroadenosine reduces levels of stabilized microtubules. A: cells were cultured for 48 h with 50 μM PE, fixed with methanol, and stained using antibodies for detyrosinated tubulin (Glu α-Tub, red), acetylated tubulin (Acetyl α-Tub, green), and total α-tubulin (α-Tub, blue), and stabilized microtubules were detected using fluorescent secondary antibodies. B: Western blot analysis of cytoskeletal α-tubulin and Glu-tubulin in confluent neonatal cardiomyocytes treated with 50 μM PE or PE + 5 μM 2-chloroadenosine. C: quantitation of Western blots (n = 6 per condition). *P ≤ 0.05 vs. control. †P < 0.05 vs. PE; **P < 0.01 vs. control; ††P < 0.01 vs. PE.

Fig. 3.

2-Chloroadenosine disrupts detyrosinated microtubules in hypertrophied neonatal rat ventricular myocytes. Cells were treated with 50 μM PE for 48 h and then for 4 h with PE in the presence or absence of 5 μM 2-chloroadenosine, 10 μM adenosine + 1 μM EHNA, or 100 nM colchicine (COLCH). Cells were stained using rabbit anti-Glu-tubulin followed by Alexa Fluor 555-labeled secondary (red) and FITC-labeled monoclonal (green) antibody to α-tubulin and Hoescht stain for DNA (blue).

Fig. 4.

Microtubules are required for cardiomyocyte hypertrophy. Cells were treated with 50 μM PE in the presence or absence of 5 μM 2-chloroadenosine or 10 μM adenosine + 1 μM EHNA for 48 h. PE-treated cells were also treated with 1 μM colchicine for 24–48 h to block polymerization of microtubules. A: cells were stained with rhodamine-phalloidin to measure cell area. B: at least 100 single cell areas were measured per condition. **P ≤ 0.01 vs. PE.

Fig. 5.

Cardiac function is impaired and microtubule (MT) stability is increased in adenosine-deficient mice. A: CD73 deletion (KO) exacerbates hypertrophy and dilation and worsens contractile function in response to transverse aortic constriction (TAC). B: fractions from ventricular homogenates of wild-type (WT) and CD73 KO mice 4 wk after sham or TAC surgery were analyzed by Western blot using antibody against total α-tubulin or Glu-tubulin. HW/BW, heart weight-to-body weight ratio; LW/BW, lung weight-to-body weight ratio. C: quantitation of Western blots. *P ≤ 0.05; **P ≤ 0.01 vs. sham control. †P < 0.05; ††P < 0.01 vs. WT animals with the same surgery. D: cold-stable Triton-insoluble tubulin levels correlate positively with congestive heart failure (as indicated by lung weight-to-body weight ratio) and inversely with ejection fraction. ▿, CD73 KO TAC; ▾, WT TAC; ○, CD73 KO sham; •, WT sham. LV, left ventricle.

Fig. 6.

Colchicine reduces pressure overload-induced accumulation of cytoskeletal tubulin. Left: after 2 wk of TAC, mice treated with saline or colchicine were killed, and heart tissue was fractionated into Triton-soluble and -insoluble (cytoskeletal) fractions and analyzed by Western blot. α-Tubulin (top) and Glu-α-tubulin (bottom) increased significantly in response to TAC, and these increases were blocked by colchicine treatment. C: quantitation of Western blot. *P ≤ 0.05; **P ≤ 0.01 vs. sham control. †P < 0.05; ††P < 0.01 vs. saline-treated animals with the same surgery.