Functional consequences of caspase activation in cardiac myocytes

Abstract

Cardiomyocyte apoptosis is present in many cardiac disease states, including heart failure and ischemic heart disease. Apoptosis is associated with the activation of caspases that mediate the cleavage of vital and structural proteins. However, the functional contribution of apoptosis to these conditions is not known. Furthermore, in cardiac myocytes, apoptosis may not be complete, allowing the cells to persist for a prolonged period within the myocardium. Therefore, we examined whether caspase-3 cleaved cardiac myofibrillar proteins and, if so, whether it affects contractile function. The effects of caspase-3 were studied in vitro on individual components of the cardiac myofilament including alpha-actin, alpha-actinin, myosin heavy chain, myosin light chain 1/2, tropomyosin, cardiac troponins (T, I, C), and the trimeric troponin complex. Exposure of the myofibrillar protein (listed above) to caspase-3 for 4 h resulted in the cleavage of alpha-actin and alpha-actinin, but not myosin heavy chain, myosin light chain 1/2, and tropomyosin, into three fragments (30, 20, and 15 kDa) and one major fragment (45 kDa), respectively. When cTnT, cTnI, and cTnC were incubated individually with caspase-3, there was no detectable cleavage. However, when the recombinant troponin complex was exposed to caspase-3, cTnT was cleaved, resulting in fragments of 25 kDa. Furthermore, rat cardiac myofilaments exposed to caspase-3 exhibited similar patterns of myofibrillar protein cleavage. Treatment with the caspase inhibitor DEVD-CHO or z-VAD-fmk abolished the cleavage. Myofilaments, isolated from adult rat ventricular myocytes after induction of apoptotic pathway by using beta-adrenergic stimulation, displayed a similar pattern of actin and TnT cleavage. Exposure of skinned fiber to caspase-3 decreased maximal Ca(2+)-activated force and myofibrillar ATPase activity. Our results indicate that caspase-3 cleaved myofibrillar proteins, resulting in an impaired force/Ca(2+) relationship and myofibrillar ATPase activity. Induction of apoptosis in cardiac cells was associated with similar cleavage of myofilaments. Therefore, activation of apoptotic pathways may lead to contractile dysfunction before cell death.

Figures

Figure 1

Cleavage of cTnT by human recombinant caspase-3. (a) Western blot, demonstrating the absence of any effect of caspase-3 on purified cTnT. (b) Western blot, demonstrating a reduction of the intact, 39-kDa cTnT and the appearance of a fragment at ≈25 kDa after caspase-3 treatment in the troponin complex. Bar graph represents mean ± SE of the percentage of the 25-kDa fragment; *, P < 0.01, caspase-3-treated compared with untreated. (c) Western blot, demonstrating a reduction of the intact, 39-kDa cTnT and the appearance of a fragment at ≈25 kDa after caspase-3 treatment when cTnT is in myofilaments. Bar graph represents mean ± SE of the percentage of the 25-kDa fragment; *, P < 0.01, caspase-3-treated compared with untreated.

Figure 2

Cleavage of cardiac α-actin by human recombinant caspase-3. (a) Silver staining of SDS/12% polyacrylamide gel, demonstrating the appearance of three bands when the monomeric purified form of α-actin is treated with caspase-3. (b) Western blot, using antibodies directed against the C terminus of α-actin, demonstrating a reduction of the intact, 42-kDa α-actin and the appearance of a major fragment at ≈15 kDa and a minor fragment at 20 kDa after caspase-3 treatment. Bar graph represents mean ± SE of the percentage of 15- and 20-kDa fragments; *, P < 0.01, caspase-3-treated compared with untreated. (c) Western blot, demonstrating a reduction of the intact, 42-kDa α-actin and the appearance of two fragments at ≈15 and 20 kDa after caspase-3 treatment of the myofilaments. Bar graph represents mean ± SE of the percentage of 15- and 20-kDa fragments; *, P < 0.01, caspase-3-treated compared with untreated.

Figure 3

Cleavage of cardiac α-actinin by human recombinant caspase-3. (a) Coomassie blue staining of SDS/7.5% polyacrylamide gel, demonstrating the appearance of one band at ≈45 kDa when the individual form of α-actinin (100 kDa) is treated with caspase-3. The other bands are contaminant proteins. (b) Western blot, demonstrating a reduction of the intact, 100-kDa α-actinin and the appearance of fragments at ≈25 kDa after caspase-3 treatment of the myofilaments.

Figure 4

Generation of cTnT and c-actin fragments during an early phase of β-adrenergic-stimulated apoptosis in ARVM. ARVM were treated with norepinephrine (NE, 10 μM) in the presence of α1-adrenergic receptor blocker prazosin (PZ, 0.1 μM) for 6 h. Where indicated, ARVM were pretreated with z-VAD-fmk (z-VAD, 100 μM). Myofilaments were isolated and analyzed by Western blotting, using antibodies anti-cTnT (a) and antibodies directed against the C-terminal region of actin (b).

Figure 5

The target site for caspase-3 on TnT. The fragment that migrates near 25 kDa displayed an N sequence of DIHRKXVEKD, indicating that it results from cTnT cleavage between Asp-96 and Asp-97. Therefore, the cleavage site for caspase-3 is DFDD. The consensus caspase-3 cleavage site usually is DEVD. The two aspartic acid residues absolutely are required, but the glutamic acid and valine residues may vary.

Figure 6

Normalized pCa tension and pCa-ATPase relation in detergent-skinned fiber bundle were measured after caspase-3 treatment. Resting tension was fixed at 2.3 μm. (a) pCa tension of untreated fiber at t = 0 (solid line) and 45 min (dashed line); maximum tensions at pCa 4.5 at t = 0 and t = 45 min were 39.6 ± 4.4 mN/mm2 and 34.0 ± 4.0 mN/mm2, respectively. (b) pCa-ATPase of untreated fiber at t = 0 and 45 min; maximum ATP consumptions at pCa 4.5 at t = 0 and t = 45 min were 335 ± 29 pmol/μl per sec and 289 ± 24 pmol/μl per sec, respectively. (c) pCa-tension of fibers after t = 0 and 45 min of caspase-3 treatment; maximum tensions at pCa 4.5 at t = 0 and t = 45 min were 37.6 ± 5.0 mN/mm2 and 16.6 ± 2.7 mN/mm2, respectively. (d) pCa-ATPase of fibers after t = 0 and 45 min of caspase-3 treatment; maximum ATP consumptions at pCa 4.5 at t = 0 and t = 45 min were 297 ± 15 pmol/μl per sec and 149 ± 15 pmol/μl per sec, respectively. (e) pCa-tension of fibers after t = 0 and 45 min of caspase-3 and DEVD-CHO (10 μM) treatments; maximum tensions at pCa 4.5 at t = 0 and t = 45 min were 36.6 ± 6.9 mN/mm2 and 27.2.0 ± 3.2 mN/mm2, respectively. (f) pCa-ATPase of fibers after t = 0 and 45 min of caspase-3 and DEVD-CHO treatments; maximum ATP consumptions at pCa 4.5 at t = 0 and t = 45 min were 312 ± 8 pmol/μl per sec and 273 ± 3 pmol/μl per sec, respectively. Graph represents mean ± SE of percent change from t = 0 min from three to four fibers, from three to four different hearts for each case. After 45 min, caspase-3 treatment decreases maximal Ca2+-activated tension as compared with sham (respectively, at pCa 4.5, caspase-3 = 39.6 ± 6.3%* vs. Sham = 82.9 ± 0.7%, P < 0.01, n = 3). Furthermore, after 45 min, caspase-3 treatment decreases maximal Ca2+-activated ATPase as compared with sham (respectively, caspase-3 = 48.4 ± 6.0%* vs. Sham = 77.9 ± 2.0%, P < 0.05, n = 3). However, there was no change in pCa50.