Adenosine kinase attenuates cardiomyocyte microtubule stabilization and protects against pressure overload-induced hypertrophy and LV dysfunction

Abstract

Adenosine exerts numerous protective actions in the heart, including attenuation of cardiac hypertrophy. Adenosine kinase (ADK) converts adenosine to adenosine monophosphate (AMP) and is the major route of myocardial adenosine metabolism, however, the impact of ADK activity on cardiac structure and function is unknown. To examine the role of ADK in cardiac homeostasis and adaptation to stress, conditional cardiomyocyte specific ADK knockout mice (cADK^-/-) were produced using the MerCreMer-lox-P system. Within 4 weeks of ADK disruption, cADK^-/- mice developed spontaneous hypertrophy and increased β-Myosin Heavy Chain expression without observable LV dysfunction. In response to 6 weeks moderate left ventricular pressure overload (transverse aortic constriction;TAC), wild type mice (WT) exhibited ~60% increase in ventricular ADK expression and developed LV hypertrophy with preserved LV function. In contrast, cADK^-/- mice exhibited significantly greater LV hypertrophy and cardiac stress marker expression (atrial natrurietic peptide and β-Myosin Heavy Chain), LV dilation, reduced LV ejection fraction and increased pulmonary congestion. ADK disruption did not decrease protein methylation, inhibit AMPK, or worsen fibrosis, but was associated with persistently elevated mTORC1 and p44/42 ERK MAP kinase signaling and a striking increase in microtubule (MT) stabilization/detyrosination. In neonatal cardiomyocytes exposed to hypertrophic stress, 2-chloroadenosine (CADO) or adenosine treatment suppressed MT detyrosination, which was reversed by ADK inhibition with iodotubercidin or ABT-702. Conversely, adenoviral over-expression of ADK augmented CADO destabilization of MTs and potentiated CADO attenuation of cardiomyocyte hypertrophy. Together, these findings indicate a novel adenosine receptor-independent role for ADK-mediated adenosine metabolism in cardiomyocyte microtubule dynamics and protection against maladaptive hypertrophy.

Keywords: Adenosine; Adenosine kinase; Cardiac hypertrophy; Detyrosinated tubulin; Microtubules.

Figure 1

Cardiomyocyte specific ADK disruption causes spontaneous LV hypertrophy. (A) Tamoxifen induced cardiomyocyte specific excision of floxed exon 7 in the ADK gene. (B) 4 weeks after the last tamoxifen injection, ADK protein expression was eliminated specifically in heart muscle in cADK^−/− mice. (C) Heart weight to body weight ratio of cADK^−/− and WT mice (n=9 WT and 10 cADK^−/− ). (D, E) β-MHC expression in WT and cADK^−/− was analyzed by western blot (n=4 WT and 4 ADK^−/−).

Figure 2. Cardiomyocyte ADK expression increases in response to LV pressure overload and attenuates pressure overload induced LV hypertrophy and fetal gene program.

(A) LV, (B) LA, and (C) RV weight to body weight ratios in WT and cADK^−/− mice after 6 weeks of control or pressure overload (TAC) conditions. (n=26, 24, 17, and 17 for WT, ADK^−/−, WT-TAC, and ADK^−/−-TAC, respectively) (D) Quantitation of fibrosis in LV sections from WT and cADK^−/− hearts (n=5, 5, 5, and 5 for WT. ADK^−/−, WT-TAC, and ADK^−/−-TAC, respectively) (E, F) WGA stain and LV cardiomyocyte cross-sectional area from WT and cADK^−/− hearts under control or TAC conditions (n=5, 5, 5, and 5 for WT. ADK^−/−, WT-TAC, and ADK^−/−-TAC, respectively) (G) Western blot and quantitation of (H), ADK (I), beta myosin heavy chain (-MHC) (J), atrial natrurietic peptide (ANP) and (K) troponin I (n=5, 5, 6, 7 for WT, ADK^−/−, WT-TAC, and ADK^−/−-TAC respectively).

Figure 3. Effects of cardiomyocyte ADK disruption on LV function during pressure overload

(A) Echocardiography measurements of end systolic diameter (ESD), (B) end diastolic diameter (EDD) and (C) ejection fraction (EF) 6 weeks after sham or TAC surgery. (n=10, 11, 12, and 14 for WT. ADK^−/−, WT-TAC, and ADK^−/−-TAC, respectively) (D) Lung weight to body weight ratio in WT and cADK^−/− mice 6 weeks after TAC (n= 26, 24, 17, and 17 for, WT, ADK^−/−, WT-TAC, and ADK^−/−-TAC, respectively). (E) Left ventricular end systolic and (F) diastolic pressures and rates of LV pressure development during (G) systole and (H) diastole in WT and cADK^−/− mice after TAC. (n=8, 9, 6, and 10 for WT. ADK^−/−, WT-TAC, and ADK^−/−-TAC, respectively)

Figure 4. Cardiac mTORC1 and p44/42 ERK MAP kinase pathways are upregulated by ADK disruption

(A-B) 6 weeks after TAC or sham surgeries, WT and cADK^−/− ventricular lysates were analyzed by western blot for phosphorylated and total levels of mTOR^Ser2448(A, B, G), p70S6k^Thr389 (A, C, H), 4E-BP^Thr37/46(A, D, I) MAP kinases ERK^{Thr202/Tyr204} (A, E, J) and p38^{Thr180/Tyr182} (A, F, K) in WT and cADK^−/− ventricular lysates under control and TAC conditions. (n=5, 5, 6, and 7 for WT. cADK^−/−, WT-TAC, and cADK^−/−-TAC respectively).

Figure 5. ADK disruption increases microtubule stabilization/detyrosination

(A) Ventricular lysates were separated into different Free, Microtubule (MT), Membrane (Memb), and Cytoskeletal (CSK) fractions as depicted. (N is nucleus, G is golgi, SR is sarcoplasmic reticulum, TT is t-tubules, M is mitochondria, SL is sarcolemma, CSK is cytoskeleton, MT is microtubule, and sMT is stabilized microtubule). Cardiac lysates were analyzed by western blot for alpha tubulin (B–F) and detyrosinated tubulin (Glu-tubulin) (G–K). β-actin (L,M), desmin (L, N) and sarcomeric actin (L,O), were also measured in the triton insoluble cytoskeletal fraction. (n=4, 4, 5, and 6 for WT. cADK^−/−, WT-TAC, and cADK^−/− TAC respectively).

Figure 6. Pharmacological inhibition of ADK reverses CADO attenuation of microtubule stabilization/detyrosination.

(A) Neonatal cardiomyocytes (NRVMs) were treated with phenylephrine (50 μM) for 48 hours in presence of CADO (5 μM), CADO + non-selective adenosine receptor antagonist 8-PT (10 μM), or CADO + ADK inhibitor iodotubercidin (ITU; 0.3 µM). α-tubulin (B and C) and detyrosinated α-tubulin (glu-tubulin) (D and E) were measured by western blot in triton soluble (sol) (B and D) and insoluble (CSK) (C and E) fractions (n ≥ 4 per condition. * indicates p < .05 compared to PE. # indicates p < .05 compared to PE-CADO ). (F) Immunofluorescence staining of NRVMs for α-tubulin and glu-tubulin after treatment described above. (G) NRVMs were treated with PE for 48 hours to induce hypertrophy, followed by an additional 24 hours with PE, PE + CADO, or PE + CADO + ABT-702(0.3 μM). α-tubulin and glu-tubulin were visualized by immunofluorescence and the area of detyrosinated tubulin was divided by the area of total tub (H) in 25–30 cells per condition. Percent change in cell area was also measured (J). Results are the average of 3 experiments, relative to continued PE treatment alone

Figure 7. ADK adenovirus augments CADO suppression of microtubule stabilization/detyrosination.

(A) Neonatal cardiomyocytes (NRVMs) were infected with β-gal or ADK expressing adenovirus (adv) for 24 hours prior to treatment with PE for 48 hours in the presence or absence of CADO (5 μM). Soluble (A, B, D) and cytoskeletal fractions (B, C, E) were examined by western blot for α-tubulin and glu-tubulin. CADO + non-selective adenosine receptor antagonist 8-PT (10 μM), or CADO + ADK inhibitor iodotubercidin (ITU; 0.3 μM). α-tubulin (B and C) and detyrosinated α-tubulin (glu-tubulin) (D and E) were measured by western blot in triton soluble (sol) (B and D) and insoluble (CSK) (C and E) fractions (n ≥ 4 per condition. * indicates p<.05 compared to PE. † indicates p<.05 compared to PE-CADO). (F) NRVMs infected with β-gal or ADK adv were examined by immunofluorescence for α-tubulin and glu-tubulin. (G) Cell area was measured in NRVMs infected with β-gal or ADK adv 48 hours after treatment with PE, PE + 1, 5, or 10 M CADO, or PE + 5 μM CADO + 0.3 μM ABT-702. (Bars represent the average cell area of at least 100 cells measured per condition; (* indicates p<.05 compared to PE. indicates p<.05 compared to PE-CADO. # indicates p<.05 comparing ADK adv to β-gal adv under same treatment)