Functional Consequences of Caspase Activation in Cardiac Myocytes

Home , Actinin, Myofilament, Troponin C, Troponin I

Functional consequences of caspase activation in cardiac myocytes

Catherine Communal*†, Marius Sumandea‡, Pieter de Tombe‡, Jagat Narula§, R. John Solaro‡, and Roger J. Hajjar*†¶

*Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129; †Harvard Medical School, Boston, MA 02115; ‡Department of Physiology and Biophysics, University of Illinois, Chicago, IL 60612; and §Division of Cardiology, Medical College of Pennsylvania͞Hanehmann, Philadelphia, PA 19102

Edited by Alexander Leaf, Massachusetts General Hospital, Charlestown, MA, and approved March 11, 2002 (received for review January 14, 2002) Cardiomyocyte apoptosis is present in many cardiac disease states, is composed of contractile proteins. Most of these proteins have including heart failure and ischemic heart disease. Apoptosis is amino acid sites amenable to specific caspase-mediated prote- associated with the activation of caspases that mediate the cleav- olysis. Because caspases are activated abundantly in the failing age of vital and structural proteins. However, the functional myocardium (1, 4, 5), it is likely that progressive cleavage of contribution of apoptosis to these conditions is not known. Fur- contractile proteins constitutes the basis of inexorable decline of thermore, in cardiac myocytes, apoptosis may not be complete, systolic ventricular function. Changes in myofilament calcium allowing the cells to persist for a prolonged period within the responsiveness and calcium-cycling proteins have been hall- myocardium. Therefore, we examined whether caspase-3 cleaved marks of experimental and human heart failure (6–10). Reduced cardiac myofibrillar proteins and, if so, whether it affects contrac- calcium sensitivity or decreased cooperation between the thick tile function. The effects of caspase-3 were studied in vitro and thin myofilaments results in reduced contractile activation on individual components of the cardiac myofilament including and force development (6). In human and experimental heart ␣-actin, ␣-actinin, myosin heavy chain, myosin light chain 1͞2, failure, several changes in the myofibrillar proteins have been tropomyosin, cardiac troponins (T, I, C), and the trimeric troponin reported to occur (7–10). These include an isoform shift in complex. Exposure of the myofibrillar protein (listed above) to troponin T (TnT) and a decrease in myosin light-chain kinase 2 caspase-3 for 4 h resulted in the cleavage of ␣-actin and ␣-actinin, (11). These changes are believed to be partly responsible for a but not myosin heavy chain, myosin light chain 1͞2, and tropo- decrease in myofibrillar ATPase activity, a decreased cross- myosin, into three fragments (30, 20, and 15 kDa) and one major bridge cycling rate, and an altered responsiveness to agents that fragment (45 kDa), respectively. When cTnT, cTnI, and cTnC were act on the myofilaments (12). incubated individually with caspase-3, there was no detectable Methods cleavage. However, when the recombinant troponin complex was exposed to caspase-3, cTnT was cleaved, resulting in fragments of In Vitro Caspase-Substrate Assay. Ten micrograms of purified 25 kDa. Furthermore, rat cardiac myofilaments exposed to cardiac myofibrillar proteins from the thick filament myosin ͞ caspase-3 exhibited similar patterns of myofibrillar protein cleav- heavy chain, myosin light chain 1 2 (from rabbit and bovine muscle, respectively; Sigma), from the thin filament cardiac age. Treatment with the caspase inhibitor DEVD-CHO or z-VAD-fmk ␣ abolished the cleavage. Myofilaments, isolated from adult rat -actin in monomer form (bovine cardiac muscle; Cytoskeleton, ventricular myocytes after induction of apoptotic pathway by Denver), tropomyosin (bovine muscle; Sigma), troponins T, I, ␤ and C (human cardiac muscle; Advanced Immunological, Long using -adrenergic stimulation, displayed a similar pattern of actin ␣ and TnT cleavage. Exposure of skinned fiber to caspase-3 de- Beach, CA), and from the cytoskeletal structure, -actinin creased maximal Ca2؉-activated force and myofibrillar ATPase (rabbit skeletal muscle; Cytoskeleton) were resuspended in caspase assay buffer (50 mM Hepes͞2 mM EDTA͞0.15 activity. Our results indicate that caspase-3 cleaved myofibrillar ͞ ͞ proteins, resulting in an impaired force͞Ca2؉ relationship and CHAPS 10% sucrose 5 mM DTT and protease inhibitors, pH 7.4). The following proteases were used to prepare the myo- myofibrillar ATPase activity. Induction of apoptosis in cardiac cells ͞ ͞ was associated with similar cleavage of myofilaments. Therefore, filaments: 1 mg ml aprotinin, 1 mg ml leupeptin, and 0.1 mM PMSF. activation of apoptotic pathways may lead to contractile dysfunc- These purified proteins then were incubated in the presence tion before cell death. or absence of recombinant human caspase-3 [protein͞caspase- 3 ϭ 500:1 (wt͞wt)for4hatroom temperature]. In a different ardiomyocyte apoptosis plays an important role in the set of experiments, troponins, reconstituted in a complexed form Cprogression of many cardiovascular disorders including (combining recombinant mouse cardiac TnT, TnI, and human heart failure. Apoptosis is mediated by caspases, specialized TnC in a ratio of 1:1:1.5) (13), were exposed to the buffer assay cysteine-dependent, aspartate-directed proteases. These pro- described above in the presence or absence of caspase-3. Cardiac teases cleave major structural elements of the cytoplasm and myofilaments, isolated from male Sprague–Dawley rat left ven- nucleus in the cells. It is being realized increasingly that apo- tricles, also were exposed to the buffer assay described above in ptosis of myocytes significantly contributes to the progressive the presence or absence of caspase-3. In the experimental set-up, loss of ventricular function in congestive heart failure (1, 2). It inhibitors of caspase-3, DEVD-CHO (10 ␮M) or z-VAD-fmk also has been demonstrated that the process of apoptosis may not [z-VAD (Z-Val-Ala-Asp(Ome)-FMK), 10 ␮M] also were added. be complete in myocytes (1, 3, 4) and may differentially affect cytoplasmic proteins and nuclear substrates (4). Lack of nuclear fragmentation facilitates continuous loss of cytoplasmic proteins This paper was submitted directly (Track II) to the PNAS office. and may allow such cells to persist for prolonged periods in Abbreviations: TnC, TnI, and TnT, troponin C, troponin I, and troponin T, respectively; myocardium. Recognition of such a process of interrupted z-VAD, Z-Val-Ala-Asp(Ome)-FMK; ARVM, adult rat ventricular myocytes. apoptosis may offer a window for reversal of myocellular damage ¶To whom reprint requests should be addressed at: Massachusetts General Hospital, Cardiovascular Research Center, 149 13th Street, CNY-4, 4215, Charlestown, MA 02129. and reverse remodeling. E-mail: [email protected]. Continued loss of cytoplasmic proteins in the presence of The publication costs of this article were defrayed in part by page charge payment. This intact nuclear integrity allows formulation of an interesting article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. hypothesis. The bulk of cytoplasmic proteins in the sarcoplasm §1734 solely to indicate this fact.

6252–6256 ͉ PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.092022999 Downloaded by guest on September 29, 2021 Poly(ADP-ribose) polymerase was used as an internal control of the activity of caspase-3. Four hours after the addition of caspase-3, the cleavage reaction was stopped by the addition of Laemmli buffer followed by sonication and heating at 95°C for 7 min. The myofilaments were spun down, and then the cleavage reaction was stopped as mentioned above.

SDS͞PAGE and Western Blot Analysis. Samples were separated on a 12% SDS-polyacrylamide gel. Gels were subjected to Coomassie blue or silver staining or transferred to nitrocellulose. Western blot analysis was achieved by using the following antibodies: anti-cTnT that recognized the C-terminal region (clone JLT-12, dilution 1:200; Sigma); two antiactin directed against C-terminal (dilution 1:200; Sigma) and N-terminal of actin, respectively (clone C4, dilution 1:1,000; Chemicon); anti-TnI (clones 10F4 and 6F9; Advanced Immunological); anti-TnC (clones 1A2, 7B9, Fig. 1. Cleavage of cTnT by human recombinant caspase-3. (a) Western blot, 12G3; Advanced Immunological); and anti-␣-actinin sarcomeric demonstrating the absence of any effect of caspase-3 on puriﬁed cTnT. (b) (clone EA-53, dilution 1:800; Sigma). The percentage of myo- Western blot, demonstrating a reduction of the intact, 39-kDa cTnT and the fibrillar protein cleavage was determined by densitometry, using appearance of a fragment at Ϸ25 kDa after caspase-3 treatment in the troponin complex. Bar graph represents mean Ϯ SE of the percentage of the NIH IMAGE. 25-kDa fragment; *, P Ͻ 0.01, caspase-3-treated compared with untreated. (c) Western blot, demonstrating a reduction of the intact, 39-kDa cTnT and the Cell Isolation, Culture, and Treatment. Adult rat ventricular myo- appearance of a fragment at Ϸ25 kDa after caspase-3 treatment when cTnT is cytes (ARVM) were isolated by enzymatic dissociation from in myoﬁlaments. Bar graph represents mean Ϯ SE of the percentage of the hearts of adult male Sprague–Dawley rats (240–280 g) as 25-kDa fragment; *, P Ͻ 0.01, caspase-3-treated compared with untreated. described. The cells were resuspended in DMEM, supplemented with albumin (2 mg͞ml), L-carnitine (2 mM), creatine (5 mM), taurine (5 mM), and 0.1% penicillin–streptomycin, and plated on regression analysis to derive the pCa50 and Hill coefficient laminin (1 ␮g͞cm2)-coated dishes. ARVM were treated for 6 h (SIGMAPLOT, Chicago). The protocol consisted of two series of with (Ϫ) norepinephrine (NE; 10 ␮M) in the presence of an measurements: a first measurement at t ϭ 0 min under control ␣ ␮ 1-adrenergic receptor blocker, prazosin (0.1 M), and ascor- conditions, followed by a second measurement after exposure of bate acid (0.1 mM) (14). In specific experiments, ARVM were the fibers to caspase-3 [ratio of protein͞caspase ϭ 1:50 (wt͞wt)] pretreated for4hwithz-VAD-fmk (100 ␮M). Myofilaments after 45 min. In sham experiments, the fibers were bathed in the then were isolated as mentioned above, and protein analysis was caspase buffer in the absence of caspase-3. The protocol was performed. repeated in the presence of the inhibitor of caspase-3, DEVD- CHO (10 ␮M). The data were calculated as percentages com- Skinned Fibers, Myofibrillar ATPase, and Forces Measurements. pared with the maximum control values obtained at t ϭ 0 min. Force͞Ca2ϩ relationships and myofibrillar ATPase activity were Cardiac myofibrillar proteins isolated from these same fibers measured simultaneously in skinned fibers by using a system also were tested for breakdown of troponins. described before (15, 16). In these experiments, fibers obtained from trabeculae of mice hearts were skinned overnight in a Statistical Analysis. Data are presented as mean Ϯ SE and were relaxing buffer (150 mM KCl͞20 mM Mops͞10 mM EGTA͞1 analyzed with a one-way ANOVA, with statistical differences ͞ ͞ ͞ Ͻ mM free Mg2 5mMATP12 mM creatine phosphate 10 identified at P 0.05. units/ml creatine kinase͞0.5 mM DTT and protease inhibitors) and 1% Triton X-100. The fibers were attached to a displace- Results ment generator at one end and a force transducer at the other Caspase-3 Activation and Myofilament Cleavage. To determine end. The resting sarcomere length was adjusted to 2.3 ␮monthe whether caspase-3 cleaves the myofilaments, we treated myofi- basis of a laser diffraction pattern. The fiber width, height, and brillar proteins individually and͞or in complexed form with length were measured, and the cross-sectional area was esti- highly purified human recombinant caspase-3 for4hand mated based on an elliptical model. Tension was expressed as analyzed the cleavage products by SDS͞PAGE and Western blot force͞cross-sectional area. To determine the relationship of free analysis. Fig. 1 demonstrates that caspase-3 induced a robust Ca2ϩ concentration to tension, the fibers were bathed sequen- cleavage of cTnT into fragments of Ϸ25 kDa only when cTnT was tially in solutions (with pCa ranging from pCa 8 to 4.5, at 20°C), in complexed form with other troponins (% of cleavage ϭ 37 Ϯ the corresponding force was measured, and the data were stored 4%, P Ͻ 0.01, as compared with control; Fig. 1b)orinthe with an analog-to-digital converter to a computer. Fiber ATPase myofilament (% of cleavage ϭ 57 Ϯ 5%, P Ͻ 0.01, as compared activity was measured as follows: ATP regeneration from ADP with control; Fig. 1c) but not individually (Fig. 1a). Of note, was coupled to the breakdown of phosphoenolpyruvate to myosin, myosin light chains, tropomyosin, TnI, and TnC were not pyruvate, and ATP was catalyzed by pyruvate kinase, which was cleaved by caspase-3. linked to the synthesis of lactate catalyzed by lactate dehydro- We then examined whether caspase-3 targets actin. Using genase. The breakdown of NADH, which is proportional to the silver staining and Western blot analyses with two different types amount of ATP consumed, was measured by using a UV light of antiactin antibodies, directed against the either the N- or MEDICAL SCIENCES that was projected through the quartz window of the bath and C-terminal region, we found that caspase-3 cleaved cardiac actin recorded at 340 nm. The ratio of light intensity at 340 nm to a in its monomeric form in two major fragments (Ϸ30 and Ϸ15 signal reference at 410 nm was obtained by the mean of an analog kDa) and two minor fragments (both Ϸ20 kDa) (Fig. 2 a and b). divider. After each recording, the UV absorbance of NADH was Caspase-3 also cleaved cardiac actin when polymerized in the calibrated by injections of ADP in the bathing solution. Data lattice of the myofilaments (Fig. 2c). The generation of the were analyzed with LABVIEW (National Instruments, Austin, Ϸ20-kDa fragments was more evident in the myofilament. TX). Data were linearized by using the Hill transformation, and In addition, Coomassie blue staining demonstrated that the force͞pCa was fitted to the Hill equation with nonlinear caspase-3 cleaved the cytoskeletal element ␣-actinin individually

Communal et al. PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 ͉ 6253 Downloaded by guest on September 29, 2021 Fig. 4. Generation of cTnT and c-actin fragments during an early phase of ␤-adrenergic-stimulated apoptosis in ARVM. ARVM were treated with norepinephrine (NE, 10 ␮M) in the presence of ␣1-adrenergic receptor blocker prazosin (PZ, 0.1 ␮M) for 6 h. Where indicated, ARVM were pretreated with z-VAD-fmk (z-VAD, 100 ␮M). Myoﬁlaments were isolated and analyzed by Western blotting, using antibodies anti-cTnT (a) and antibodies directed against the C-terminal region of actin (b).

Fig. 2. Cleavage of cardiac ␣-actin by human recombinant caspase-3. (a) ͞ kDa. Cleavages of cTnT and actin were reduced after pretreat- Silver staining of SDS 12% polyacrylamide gel, demonstrating the appear- ␤ ance of three bands when the monomeric puriﬁed form of ␣-actin is treated ment with VAD-fmk (Fig. 4), demonstrating that -adrenergic with caspase-3. (b) Western blot, using antibodies directed against the C receptor-stimulated myofilament cleavage occurred mainly via terminus of ␣-actin, demonstrating a reduction of the intact, 42-kDa ␣-actin caspase activation. and the appearance of a major fragment at Ϸ15 kDa and a minor fragment at 20 kDa after caspase-3 treatment. Bar graph represents mean Ϯ SE of the Cleavage Site on TnT. To determine the major site of caspase-3 percentage of 15- and 20-kDa fragments; *, P Ͻ 0.01, caspase-3-treated cleavage of cTnT, caspase-3-generated cTnT fragments from the compared with untreated. (c) Western blot, demonstrating a reduction of the troponin complex were isolated from a polyvinylidene mem- intact, 42-kDa ␣-actin and the appearance of two fragments at Ϸ15 and 20 kDa after caspase-3 treatment of the myoﬁlaments. Bar graph represents brane, and partial amino acid sequences were obtained by mean Ϯ SE of the percentage of 15- and 20-kDa fragments; *, P Ͻ 0.01, Edman degradation and HPLC. The fragment that migrated caspase-3-treated compared with untreated. near 25 kDa displayed an N sequence of DIHRKXVEKD, indicating that it did, in fact, result from cTnT cleavage between Asp-96 and Asp-97 as shown in Fig. 5. This finding points to the in fragments Ϸ45 kDa. This was blocked by the addition of cleavage site for caspase-3 as being DFDD. The consensus caspase inhibitor z-VAD (Fig. 3a). Furthermore, caspase-3 also caspase-3 cleavage site is usually DEVD. The two aspartic acid cleaved ␣-actinin in the lattice of the myofilaments. A fragment residues are absolutely required, but the glutamic acid and valine of Ϸ25 kDa was detected by using Western blot analysis (Fig. 3b). residues may vary. Comparison between cardiac TnT from mice, The addition of capase-3 inhibitors also blocked the cleavage of rat, and human revealed that this site is highly conserved. cTnT, cardiac ␣-actin (data not shown). Because we had no access to antibodies directed against the N-term of cTnT, we treated a troponin complex (cMyc attached Effect of ␤-Adrenergic Stimulation on Myofilament Breakdown. We to cTnT) with caspase-3, and, after treatment, we isolated the have shown that ␤-adrenergic receptor stimulation induces troponin complex and probed with antibodies directed against apoptosis in adult rat ventricular myocytes, and this was asso- cMyc. We could not find the cMyc fragment (N-terminal frag- ciated with the early activation of the mitochondrial pathway and ment), suggesting that only the C terminus remains in the activation of caspase-3. Therefore, ARVM were exposed to complex or in the lattice of the myofilament. This N-terminal ␤-adrenergic stimulation for 6 h and myofilaments then were region has been shown to be important for the cardiac function. isolated during the early phase of apoptosis when no major DNA Indeed, the truncation of the N-terminal region of cTnT impairs breakdown (terminal deoxynucleotidyltransferase-mediated the force͞Ca2ϩ relationship and decreases myofibrillar ATPase UTP end labeling-positive cells) is found. ␤-Adrenergic receptor activity in skinned fiber. stimulation induced similar cleavage of cTnT (Ϸ25 kDa) in ARVM (Fig. 4). The antibodies against the N-terminal of actin Caspase Activation and Contractile Activation. To examine whether (residues 23–24) and C-terminal (last 11 residues) recognized, in the activation of caspase-3 and the concomitant breakdown of addition to the intact actin, a similar, major fragment of Ϸ20 cardiac TnT, ␣-actin, and ␣-actinin result in functional effects, we measured the effect of caspase-3 treatment on force͞Ca2ϩ relationship and simultaneous myofibrillar ATPase activity in skinned fibers isolated from the mice hearts. Fig. 6 illustrates the force͞Ca2ϩ relationship and simultaneous myofibrillar ATPase before (t ϭ 0) and after 45 min of caspase-3 treatment. After 45

Fig. 3. Cleavage of cardiac ␣-actinin by human recombinant caspase-3. (a) Coomassie blue staining of SDS͞7.5% polyacrylamide gel, demonstrating the Fig. 5. The target site for caspase-3 on TnT. The fragment that migrates near appearance of one band at Ϸ45 kDa when the individual form of ␣-actinin 25 kDa displayed an N sequence of DIHRKXVEKD, indicating that it results (100 kDa) is treated with caspase-3. The other bands are contaminant proteins. from cTnT cleavage between Asp-96 and Asp-97. Therefore, the cleavage site (b) Western blot, demonstrating a reduction of the intact, 100-kDa ␣-actinin for caspase-3 is DFDD. The consensus caspase-3 cleavage site usually is DEVD. and the appearance of fragments at Ϸ25 kDa after caspase-3 treatment of the The two aspartic acid residues absolutely are required, but the glutamic acid myoﬁlaments. and valine residues may vary.

6254 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.092022999 Communal et al. Downloaded by guest on September 29, 2021 caspase-3 (Fig. 6 e and f) but DEVD-CHO had no independent effects on myofilament activation (data not shown). To ascertain that these fibers, which were used for the mechanical studies, indeed had breakdown of cardiac TnT, actin, and ␣-actinin, we analyzed these fibers by Western blotting. As in the myofilaments, we found one detectable cleavage product for cTnT (at 25 kDa), two detectable products for actin (20 and 15 kDa), and one cleavage product for ␣-actinin (at 25 kDa) (data not shown). Discussion Our results demonstrate that caspase-3 activation directly targets three components of the myofilaments, namely, ␣-actin, ␣-actinin, and TnT. Interestingly, TnT was cleaved only when it was combined with the other troponins. Furthermore, pro-apoptotic stimuli such as ␤-adrenergic stimulation induce similar break- downs of myofilaments. Finally, the cleavage of these proteins by caspase-3 had direct functional effects on myofilament activation and contractile function. In cardiac tissue, apoptosis is a form of cell death that has been described as distinct from necrotic cell death. It has been proposed that ventricular dilatation and neurohormonal activation during heart failure lead to up-regulation of transcription factors, induce myocyte hypertrophy, and prepare the cell for entry into the cell cycle. However, terminally differentiated myocytes cannot divide, and, instead, they undergo apoptosis. Initiation of apoptosis is associated with activation of upstream cascades, including the release of cytochrome c from mitochon- dria to cytoplasm and the processing of proteolytic caspases. The activation of caspases leads to fragmentation of various cytoplasmic proteins. However, the nuclear fragmentation and con- densation is completed only rarely. The balance between nuclear fragmentation and protein cleavage is variable in myocytes (1, 3, 4). When this balance is shifted toward the continuous loss of cytoplasmic proteins, cells persist but, in the case of cardiomy- Fig. 6. Normalized pCa tension and pCa-ATPase relation in detergent- ocytes, are in a ‘‘Zombie’’ state with little or no contraction. skinned fiber bundle were measured after caspase-3 treatment. Resting ten- When this balance is shifted toward nuclear fragmentation, the sion was fixed at 2.3 ␮m. (a) pCa tension of untreated fiber at t ϭ 0 (solid line) cells die. This state of interrupted apoptosis, where cardiac and 45 min (dashed line); maximum tensions at pCa 4.5 at t ϭ 0 and t ϭ 45 min myocytes are dysfunctional (without nuclear fragmentation), were 39.6 Ϯ 4.4 mN͞mm2 and 34.0 Ϯ 4.0 mN͞mm2, respectively. (b) pCa- may offer a window for reversal of myocellular damage and ATPase of untreated fiber at t ϭ 0 and 45 min; maximum ATP consumptions reverse remodeling. at pCa 4.5 at t ϭ 0 and t ϭ 45 min were 335 Ϯ 29 pmol͞␮l per sec and 289 Ϯ Our results would suggest that the activation of caspases 24 pmol͞␮l per sec, respectively. (c) pCa-tension of fibers after t ϭ 0 and 45 min ϭ ϭ indeed induce the breakdown of myofibrillar proteins, which of caspase-3 treatment; maximum tensions at pCa 4.5 at t 0 and t 45 min leads to a decrease in ATPase activity and force development. were 37.6 Ϯ 5.0 mN͞mm2 and 16.6 Ϯ 2.7 mN͞mm2, respectively. (d) pCa- ATPase of fibers after t ϭ 0 and 45 min of caspase-3 treatment; maximum ATP This decrease in systolic function therefore could precede any consumptions at pCa 4.5 at t ϭ 0 and t ϭ 45 min were 297 Ϯ 15 pmol͞␮l per breakdown of DNA. The cleavage of the myofilaments clearly sec and 149 Ϯ 15 pmol͞␮l per sec, respectively. (e) pCa-tension of fibers after results in a reduction of systolic function, which can be detri- t ϭ 0 and 45 min of caspase-3 and DEVD-CHO (10 ␮M) treatments; maximum mental for the overall function of the heart. Interestingly, in a tensions at pCa 4.5 at t ϭ 0 and t ϭ 45 min were 36.6 Ϯ 6.9 mN͞mm2 and model of ischemia reperfusion, blockade of apoptosis by gene 27.2.0 Ϯ 3.2 mN͞mm2, respectively. (f) pCa-ATPase of fibers after t ϭ 0 and 45 transfer of akt improved ventricular wall shortening. However, it min of caspase-3 and DEVD-CHO treatments; maximum ATP consumptions at also could serve to decrease energy utilization while the cell is pCa 4.5 at t ϭ 0 and t ϭ 45 min were 312 Ϯ 8 pmol͞␮l per sec and 273 Ϯ 3 ͞␮ Ϯ attempting to actively repair DNA, a costly endeavor for the cell pmol l per sec, respectively. Graph represents mean SE of percent change from an energy standpoint. from t ϭ 0 min from three to four fibers, from three to four different hearts for each case. After 45 min, caspase-3 treatment decreases maximal Ca2ϩ- Structurally, cardiac troponin T connects tropomyosin to the activated tension as compared with sham (respectively, at pCa 4.5, caspase-3 ϭ TnC–TnI complex and confers calcium sensitivity and cooper- 39.6 Ϯ 6.3%* vs. Sham ϭ 82.9 Ϯ 0.7%, P Ͻ 0.01, n ϭ 3). Furthermore, after 45 ativity to the activity of the TnC–TnI complex on actomyosin min, caspase-3 treatment decreases maximal Ca2ϩ-activated ATPase as com- ATPase (6, 15–18). The C terminus of TnT is necessary for pared with sham (respectively, caspase-3 ϭ 48.4 Ϯ 6.0%* vs. Sham ϭ 77.9 Ϯ submaximal activation whereas the N terminus is essential for 2.0%, P Ͻ 0.05, n ϭ 3). However, there was no change in pCa50. maximal activation. In our studies, cleavage of TnT occurred only when it was complexed, probably because the cleavage site MEDICAL SCIENCES is exposed in that complex. A number of TnT mutations (mis- min of caspase-3 treatment, there was a significant decrease in sense and base pair deletions) have been associated with hyper- 2ϩ 2ϩ maximal Ca -activated force and in Ca sensitivity (Fig. 6c)as trophic cardiomyopathies (19, 20). Functionally, these TnT compared with sham fibers that were treated with buffer solution ϩ mutations result in decreases in maximal activated force, with (Fig. 6a). There was a parallel decrease in maximal Ca2 - variable effects on calcium sensitivity. Interestingly, our results activated myofibrillar ATPase activity (Fig. 6d) as compared revealed that the maximal Ca2ϩ activation force was affected with sham fiber (Fig. 6b). The addition of caspase-3 inhibitor without much change in calcium sensitivity. A recent study DEVD-CHO prevented the functional changes mediated by showed that after ischemia͞reperfusion, caspase inhibition

Communal et al. PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 ͉ 6255 Downloaded by guest on September 29, 2021 blocked the breakdown of TnI (21). These results would seem at Interestingly, we did not find any breakdown of the other odds with our data, wherein we did not observe breakdown of troponins with activation of caspase-3. Furthermore, myosin TnI in the presence of caspase-3. However, in their study, molecule also was unaffected by the activation of caspase-3. This Ruetten et al. (21) found that the breakdown of TnI depended could be because there is a small amount of degradation and directly on calpain activation, which was not the case with our because our methods of detection are not sensitive enough. preparations. Furthermore, stunning in ischemia reperfusion These results show an important interrelationship between specifically induces TnI (22), which may not be preponderant in other cardiovascular conditions and diseases in which cardiac caspase activation and functional reserve in cardiomyocytes. apoptosis is present. Targeting caspases in cardiomyocytes may be beneficial not only Another target of caspase-3 was ␣-actinin, which is a main for cardiomyocyte survival but also function. component of the Z band and is responsible for linking the thin filaments together (23). The role of ␣-actinin has been proposed This work was supported in part by grants from the National Institutes to keep the thin filaments in a cooperative mode during con- of Health [HL 57623 to R.J.H.; R01-63704 to P.d.T.; and PO1-HL62426 traction and relaxation. In ischemia, the loss of ␣-actinin has (project 1 to R.J.S. and M.S. and project 4 to P.d.T.)]; and a Doris Duke been thought to decrease maximal force of contraction by Charitable Foundation Clinician Scientist Award (to R.J.H.). R.J.H. is altering the transmission of force from the actin–myosin reaction a Beeson Scholar of the American Federation of Aging Research. C.C. and by altering interfilament spacing. These effects would cause was supported in part by Grant HL61557 to Dr. Anthony Rosenzweig. a decrease in the affinity of actin for ␣-actinin and disrupt M.S. is supported in part by an Individual National Research Service parallel interfilament formation. Award (F32HL10409–02).

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6256 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.092022999 Communal et al. Downloaded by guest on September 29, 2021