We have previously indicated that calpain inhibitor-1 prevents the heart from ischemia- reperfusion injury associated with the impairment of total Ca2+ handling by inhibiting the proteolysis of α-fodrin. However, this inhibitor is insoluble with water and inappropriate for clinical application. The aim of the present study was to investigate the protective effect of a newly developed calpain inhibitor, SNJ-1945 (SNJ), with good aqueous solubility on left ventricular (LV) mechanical work and energetics in the cross-circulated rat hearts. SNJ (150 μM) was added to KCl (30 meq) cardioplegia (CP). Mean end-systolic pressure at midrange LV volume (ESPmLVV) and systolic pressure-volume area (PVA) at mLVV (PVAmLVV; a total mechanical energy per beat) were hardly changed after CP plus SNJ arrest-reperfusion (post-CP + SNJ), whereas ESPmLVV and PVAmLVV in post-CP group were significantly (P < 0.01) decreased. Mean myocardial oxygen consumption for the total Ca2+ handling in excitation-contraction coupling did not significantly decrease in post-CP + SNJ group, whereas it was significantly (P < 0.01) decreased in post-CP group. The mean amounts of 145- and 150-kDa fragments of α-fodrin in the post-CP group were significantly larger than those in normal and post-CP + SNJ groups. In contrast, the mean amounts of L-type Ca2+ channel and sarcoplasmic reticulum Ca2+-ATPase were not significantly different among normal, post-CP, and post-CP + SNJ groups. Our results indicate that soluble SNJ attenuates cardiac dysfunction due to CP arrest-reperfusion injury associated with the impairment of the total Ca2+ handling in excitation-contraction coupling by inhibiting the proteolysis of α-fodrin.
- cardiac function
- systolic pressure-volume area
- oxygen consumption
it is well known that one of underlying mechanisms for ischemia-reperfusion (I/R) injury is Ca2+ overload resulting from increased Ca2+ influx mediated via reverse-mode Na+-Ca2+ exchanger (NCX) (8a, 12, 27, 34). Our laboratory has previously reported that reperfusion injury after KCl cardioplegic cardiac arrest leads to Ca2+ overload and ventricular dysfunction similar to I/R injury (13). The mechanisms of Ca2+ overload in this model likely involve an accumulation of intracellular Na+ and subsequent activation of reverse mode NCX activity (25). However, previous studies in our laboratory suggest that proteolysis of the cytoskeletal protein α-fodrin by calpains may also play a role (13, 37, 42).
Calpain inhibition was found to prevent against the proteolysis of α-fodrin due to reperfusion injury after global ischemia (42). It has been proposed that α-fodrin maintains the integrity of the plasma membranes as a constituent of the membrane skeleton (3, 17). Therefore, it seems likely that the degradation of α-fodrin in membrane fractions would alter the properties of ion channels (40). Indeed, the possibility that disruption of cytoskeletal proteins inactivates L-type Ca2+ channels has been reported (8, 22).
We hypothesized that a novel calpain inhibitor, ((1S)-1((((1S)-1-benzyl-3-cyclopropyl- amino-2,3-di-oxopropyl) amino)carbonyl)-3-methylbutyl) carbamic acid 5-methoxy-3oxapentyl ester (SNJ-1945; SNJ) would attenuate left ventricular (LV) dysfunction following cardioplegic cardiac arrest by inhibiting the proteolysis of α-fodrin. This novel calpain inhibitor (SNJ) has been shown to have good aqueous solubility, good plasma exposure, and good tissue penetration in rats and monkeys (24, 29). Furthermore, intraperitoneal administration of SNJ (160 mg/kg) for 14 days produced no obvious toxicity or abnormalities in rats (24). SNJ also exerted inhibitory actions on Ca2+-independent proteinase and cathepsin L and B (personal communication from M. Yamaguchi). Furthermore, it was reported that SNJ was effective against cerebral ischemia-induced damage (14), but there have been no reports on its efficacy against cardiac ischemia. The aim of the present study was to investigate the cardioprotective effects of SNJ added in KCl cardioplegia against reperfusion injury after cardioplegic cardiac arrest in a cross-circulated rat heart model using analysis of LV mechanical work and energetics. This blood-perfused model offers a closer approximation to the clinical scenario than crystalloid ones, although care should be taken in extrapolating results from the present study to the clinical setting.
The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). All surgical and experimental protocols were approved by and performed according to the guidelines for the care and use of animals established by Nara Medical University.
Experiments were performed on 24 excised, cross-circulated rat heart preparations, as reported previously (8a, 9, 10, 37, 38, 42). In each experiment, three retired breeder male crj: Wistar rats weighing 641 ± 90 g (16–25 wk of age; n = 72), purchased from Charles River Japan (Yokohama, Japan), were anesthetized with pentobarbital sodium (50 mg/kg ip) and intubated for artificial ventilation. All rats were heparinized (1,000 units IV). The largest rat was used as a blood supplier to extract its blood for priming the cross-circulation tubing. The chest was opened midsternally, and the blood was drained via a 21-gauge needle stabbed into the LV. The middle-size rat was used as the metabolic supporter; the bilateral common carotid arteries and right external jugular vein were cannulated with the arterial and venous cross-circulation tubing, respectively. The chest of the smallest rat as the heart donor was opened midsternally under artificial ventilation. The brachiocephalic artery and the right ventricle via the superior vena cava were cannulated and connected to the arterial and venous cross-circulation tubing from the support rat. The heart-lung section was isolated from the left common carotid artery, descending aorta, inferior vena cava, and pulmonary trunk in this order. The beating heart, supported by cross-circulation, was then excised from the chest. Coronary perfusion of the excised heart was never interrupted during the preparation (Fig. 1) (9). The excised heart was maintained at 37°C.
A thin latex balloon (balloon material volume, 0.08 ml) fitted into the LV was connected to a pressure transducer (Life Kit DX-312; Nihon Kohden, Tokyo, Japan) and a 0.5-ml precision glass syringe with fine scales (minimum scale, 0.005 ml). The maximum unstretched balloon volume was below ∼0.20–0.25 ml. Thus LV volume (LVV) was changed and measured by adjusting the intraballoon water volume with the syringe in 0.05-ml steps between 0.08 and 0.23 ml.
Systolic unstressed volume (V0) was determined by filling the balloon to the level where peak isovolumic pressure and hence pressure-volume area (PVA; see Data analysis) were zero. The sum of intraballoon water volume and balloon material volume (0.08 ml) was used as an initial estimate of V0. This procedure was repeated during different LVV loading runs. V0 was then finally determined as the volume-axis intercept of the best-fit end-systolic pressure-volume relationship (ESPVR). We obtained the best-fit ESPVR with Eq. 1 (see Table 1) by means of the least-squares method (Delta-Graph; DeltaPoint, Monterey, CA) on a personal computer (1, 9, 10, 23, 37, 38). Correlation coefficients of the best-fit ESPVRs were higher than 0.99 (Table 1).
The LV epicardial electrocardiogram was recorded, and the heart rate was constantly maintained by electrical pacing of the right atrium. The pacing rate (=300 beats/min) was adjusted to avoid causing incomplete relaxation or arrhythmia. The systemic arterial blood pressure of the supporter rat served as the coronary perfusion pressure (100–130 mmHg; Fig. 1). Arterial pH, Po2, and Pco2 of the supporter rat were maintained within their physiological ranges with supplemental O2 and sodium bicarbonate. To do this, we checked arterial pH, Po2, and Pco2 of the supporter rat by arterial blood sampling every half an hour.
Total coronary blood flow was continuously measured with an electromagnetic flowmeter (MFV-3100; Nihon Kohden, Tokyo, Japan) placed in the middle of the coronary venous drainage tubing from the right ventricle. LV thebesian flow was negligible. The coronary arteriovenous O2 content difference (AVO2D) was continuously measured by passing all the arterial and venous cross-circulation blood through the cuvettes of a custom-made oximeter (PWA-200S; Shoe Technica, Chiba, Japan) as previously reported in detail (Fig. 1) (9, 37, 38). The mean concentration of hemoglobin in the perfused blood was 15.8 ± 1.8 mg/dl.
We attempted to fit experimentally obtained LV pressure-volume data to the exponential equations to obtain ESPVRs (see Table 1) and end-diastolic pressure-volume relationships (EDPVRs). PVA is defined as the pressure-volume area circumscribed by the curvilinear best-fit ESPVRs, EDPVRs, and the systolic portions of the ventricular pressure-volume trajectories at any LVVs. The area under the EDPVR was reasonably assumed to be zero within the same volume range. Finally, PVA was normalized by LV mass to 1 g. Based on our previous proposal (9, 10, 18, 31, 38), mean end-systolic pressure at midrange LV volume (ESPmLVV) and systolic PVA at mLVV (PVAmLVV; a total mechanical energy per beat) were calculated to assess LV mechanical work and energetics.
As shown previously (1, 9, 37, 42), the V̇o2-PVA relationship was linear in the rat LV. Its slope represents the O2 cost of PVA, and its V̇o2 intercept represents the PVA-independent V̇o2. The PVA-independent V̇o2 is composed of O2 consumption for Ca2+ handling in excitation-contraction (E-C) coupling (33) and for basal metabolism (23, 32). The right ventricle was kept collapsed by continuous hydrostatic drainage of the coronary venous return so that the right ventricular PVA and hence PVA-dependent V̇o2 were assumed to be negligible (9, 38). The right ventricular PVA-independent V̇o2 (1, 9, 37, 42) was subtracted from the total V̇o2 to yield LV V̇o2. The LV (including the septum) and the right ventricle were weighed for standardization of LVV. They were 1.07 ± 0.12 and 0.32 ± 0.06 g (n = 8) in 30 meq KCl containing cardioplegic cardiac arrest (CP) group, 1.25 ± 0.08 and 0.36 ± 0.07 g (n = 8) in the novel calpain inhibitor SNJ containing cardioplegic cardiac arrest (CP + SNJ) group and 1.05 ± 0.15 and 0.30 ± 0.05 g (n = 8) in normal group.
LV pressure (LVP), V̇o2, and PVA data during isovolumic contractions were simultaneously obtained at five to six different LVVs (EDVs) in each one heart (vol-run). All hearts underwent 30-min cardioplegic cardiac arrest (37°C) and 60-min blood reperfusion. Cardiac arrest was obtained for 3 min by administration of cardioplegic solution through the arterial perfusion tube (Fig. 1, arrow No. 2), and, simultaneously, 3 ml of the blood stayed at the coronary circulation system was removed from the venous return tube (Fig. 1, arrow No. 5); the following cardioplegic cardiac arrest was employed for 27 min (Figs. 1 and 2). The concentration of KCl cardioplegic solution was determined as 30 meq by previous studies (13, 16, 19). The concentration of SNJ added to cardioplegic solution was determined as 150 μmol/l. This concentration is about fourfold effective cardioprotective concentration (40 μM; this concentration was determined by preliminary studies) to avoid any inactivation of SNJ during the 30-min cardioplegic cardiac arrest (37°C). At the onset of reperfusion, 3 ml of the blood perfused into the coronary circulation system was removed from the venous return tube (Fig. 1, arrow No. 5). After 60-min reperfusion with and without infusion of 40 μmol/l SNJ, the vol-runs were performed again (post-CP and post-CP + SNJ vol-runs; Fig. 2A, protocols A and B). Coronary flow was monitored during the protocol (Fig. 2B). Although protocol C was shorter than protocols A and B, we have confirmed that LV mechanoenergetic data obtained by normal vol- and Ca2+ ino-runs were constant during 4 to 5 h after onset cross-circulation (data not shown; Fig. 2A).
After the post-CP and post-CP + SNJ vol-runs, a Ca2+-induced different inotropic run (Ca2+ ino-run) was performed at mLVV [0.16 ml = 0.08 ml (water volume infused into the balloon) plus 0.08 ml (V0)] during intracoronary infusion of 1% CaCl2 solution. The infusion rate of Ca2+ was increased in steps up to 1–20 ml/h (n = 8 in each group). In every vol-run and ino-run, LVP, AVO2D, and coronary flow (CF) were stable 3 min after changing LVV and infusion rate of Ca2+.
To measure basal metabolic O2 consumption, cardiac arrest was induced by infusing KCl (0.5 mol/l) into the coronary perfusion tubing at 10 ml/h (n = 3 each) that was adjusted to abolish electrical excitation under monitoring ventricular electrocardiograms but not to generate any KCl-induced constrictions of coronary vessels. V̇o2 and PVA data were obtained by minimal volume loading to avoid volume-loading effects on V̇o2 data. In each steady state, data were sampled at 500 Hz for 3 s, and sampling was usually repeated three times at intervals of 0.5–1 min.
A synthetic calpain inhibitor SNJ (provided from Senju Pharmaceutical) was dissolved in Lactate Ringer solution at 600 μmol/l. We adjusted the blood concentration of SNJ at 40 μmol/l by changing the infusion rate (4.0–14.6 ml/h) under monitoring the real-time CF. Intracoronary infusion of SNJ was continued for 60 min during reperfusion (n = 8). Mean values for mLVV and V0 (normalized for LV 1 g) were 0.15 ± 0.03 ml/g and 0.08 ± 0.01 ml/g, respectively (n = 24).
Oxygen cost of LV contractility.
We obtained the specific best-fit curve for the observed ESPmLVV and ESP (0 mmHg at V0) with the best-fit ESPVR function in post-CP or post-CP + SNJ vol-run by the least-squares method, and we calculated PVAmLVV during Ca2+ infusion using this specific best-fit curve function on a personal computer (26, 30, 37, 38). The parallelism of the V̇o2-PVA linear relation during Ca2+ infusion has been confirmed in normal hearts (30) and was also confirmed in the present post-CP and post-CP + SNJ hearts (n = 3). From this parallelism, the lines including all V̇o2-PVA data obtained during Ca2+ infusion in steps at mLVV were drawn in parallel to the V̇o2-PVA relation line in post-CP or post-CP + SNJ vol-run before Ca2+ infusion, as described previously (9, 26, 30, 38). The gradually increased V̇o2-intercept values (PVA-independent V̇o2 values) of the lines proportional to the enhanced LV contractility by Ca2+ were obtained by this procedure.
Our proposed index for LV contractility, equivalent maximal elastance (eEmax), was calculated from a triangular area equivalent to PVAmLVV (30, 38). The O2 cost of LV contractility was the slope of the relationship between PVA-independent V̇o2 and eEmax, i.e., V̇o2 used for Ca2+ handling in E-C coupling per unit changes in LV contractility (30, 38).
Analyses of one-beat LV pressure-time curve by hybrid and single logistic functions.
To evaluate the LV systolic and diastolic functions, we analyzed the contraction rate (b corresponds to change in maximal change in pressure over time +dP/dtmax) and relaxation rate (i corresponds to minimal change pressure over time −dP/dtmax) from a best-fit function to one-beat LV pressure-time curve at mLVV during contraction and relaxation with our proposed hybrid logistic function (20) in normal, post-CP, and post-CP + SNJ group hearts (n = 8 each). To evaluate LV end-diastolic relaxation rate or lusitropism, we analyzed logistic time constant from respective best-fit functions to one-beat LV pressure-time curve at mLVV during relaxation with our proposed single logistic function (21) in normal, post-CP and post-CP + SNJ group hearts (n = 8 each).
PAGE and immunoblottings of 150-kDa and 145-kDa fragments of α-fodrin (250-kDa), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and L-type Ca2+ channel.
We have previously reported that proteolysis of a cytoskeleton protein, α-fodrin, is found without proteolysis of ankyrin, connexin43, and troponin I in high Ca2+ infusion-induced Ca2+ overloaded contractile failing hearts associated with the impairment of the total Ca2+ handling in the E-C coupling (37). It seems likely that α-fodrin is the most sensitive membrane protein to Ca2+ overload. To compare cardiac protective effect of KCl cardioplegia (CP) with that of CP + SNJ, we focused on proteolysis of α-fodrin in the present cardioplegic arrest-reperfusion injury, because there is a close correlation between the membrane α-fodrin proteolysis and the impairment of the total Ca2+ handling, where the V̇o2 intercept was decreased, basal metabolic O2 consumption was unaltered and oxygen costs of PVA and LV contractility were unaltered (32, 37, 42).
In addition, recent studies have revealed that calpain activation induced by I/R injury causes degradation of Ca2+-handling proteins such as L-type Ca2+ channel (LTCC)(6) and SERCA2a (7). Thus we also examined the degradation of Ca2+-handling proteins such as LTCC and SERCA2a.
Membrane proteins were isolated from the LV wall of each frozen heart stored at −80°C after the mechanoenergetic studies. The frozen hearts were homogenized and centrifuged at 1,000 g for 10 min. The supernatants were centrifuged at 100,000 g for 60 min at 4°C. The 100,000 g pellets were cellular membrane fractions and used for immunoblotting of 150- and 145-kDa fragment of α-fodrin (240-kDa)(35, 41, 42), SERCA2a, and LTCC. The membranes were blocked (4% Block Ace; Dainippon Pharmaceutical, Osaka) and then incubated with 2,000-fold diluted primary antibody against anti-α-fodrin (1:2,000 dilution; Biohit, Genex), anti-SERCA2a antibody (1:1,000 dilution; Affinity Bio Reagents), and anti-LTCC antibody (1:300 dilution; Alomone). The antigens were detected by the luminescence method (ECL Western blotting detection kit; Amersham) with peroxidase-linked anti-mouse IgG (1:2,000 dilution) or peroxidase-linked anti-rabbit IgG (1:2,000). The amounts of membrane proteins were determined to obtain the linear response of ECL immunoblot. After immunoblotting, the film was scanned with a scanner, and the intensity of the bands was calculated by NIH image analysis. The intensity ratio of 145- and 150-kDa bands versus 240-kDa band (α-fodrin) was expressed in an arbitrary unit. The statistical difference was evaluated by ANOVA with Bonferroni's post hoc analysis.
Comparison of paired and unpaired individual values was performed by paired and unpaired t-test, respectively. Multiple comparisons were performed by one-way ANOVA with post hoc Bonferroni's test. A value of P < 0.05 was considered statistically significant. All data are expressed as the means ± SD.
LV mechanoenergetics after I/R with and without infusion of SNJ.
Figure 3 showed each representative set of ESPVRs and EDPVRs and V̇o2-PVA relations in pre- and post-CP or pre- and post-CP + SNJ. Curvilinear ESPVR in post-CP markedly shifted downward (Fig. 3A). Each PVA and V̇o2 value at each LVV (EDV) in post-CP was markedly smaller than each pre-CP value, and the V̇o2-PVA linear relation in post-CP shifted downward without changes in the slope (Fig. 3B). On the other hand, each set of ESPVR and EDPVR in post-CP + SNJ hardly changed (Fig. 3C), and V̇o2-PVA linear relation in post-CP + SNJ also hardly changed; the V̇o2-PVA relations in pre- and post-CP + SNJ could be superimposed; neither slope nor V̇o2 intercept changed (Fig. 3D).
Summarized data of LV mechanics and energetics are shown in Table 1 and Figs. 4 and 5. Mean ESPmLVV was significantly (P < 0.01) decreased in the post-CP group (Fig. 4A), whereas it was unchanged in post-CP + SNJ group (Fig. 4C). Mean PVAmLVV was significantly (P < 0.01) decreased in the post-CP group (Fig. 4B), whereas it was unchanged in post-CP + SNJ group (Fig. 4D).
Mean slopes of V̇o2-PVA linear relations in post-CP and post-CP + SNJ groups were unchanged compared with pre-CP and pre-CP + SNJ groups, respectively (Fig. 5, A and C), indicating no significant differences in PVA-dependent V̇o2 in any PVA between pre- and post-CP (see Fig. 3B), and between pre- and post-CP + SNJ (see Fig. 3D). Mean V̇o2-intercept value (PVA-independent V̇o2) of V̇o2-PVA linear relation was significantly (P < 0.01) decreased in the post-CP group (Fig. 5B), whereas it was unchanged in the post-CP + SNJ group (Fig. 5D).
Mean ESPmLVV and PVAmLVV in post-CP group were decreased to 69.9 ± 7.6% of pre-CP and 68.7 ± 10.2% of pre-CP, respectively. SNJ significantly (P < 0.01) antagonized CP-induced decrease in mean ESPmLVV and PVAmLVV to 97.8 ± 5.1% of pre-CP + SNJ and 102.8 ± 8.6% of pre-CP + SNJ, respectively (Fig. 6, A and B). Mean V̇o2 intercept in the post-CP group was decreased to 60.6 ± 6.6% of pre-CP. SNJ significantly (P < 0.01) antagonized CP-induced decrease in mean V̇o2 intercept to 97.7 ± 13.1% of pre-CP + SNJ (Fig. 6C).
Basal metabolic O2 consumption was unaltered in the post-CP and post-CP + SNJ groups compared with that in normal hearts (post-CP, 31.2 ± 8.2; post-CP + SNJ, 29.2 ± 5.4; normal, 30.4 ± 10.1 μlO2·min−1·g−1). Therefore, the decreased V̇o2 intercept means the decreased V̇o2 utilized for Ca2+ handling in E-C coupling.
Immunoblotting of 150-kDa and 145-kDa fragments of α-fodrin (240-kDa).
Figure 7 showed immunoblottings of 240 kDa α-fodrin and 145- and 150-kDa α-fodrin proteolytic fragments in each group. The mean amounts of 145- and 150- kDa fragments in the post-CP group were significantly larger than those in normal and post-CP + SNJ groups. There was no significant difference in mean amounts of α-fodrin proteolytic fragments between normal and post-CP + SNJ groups. Percent decreases in ESPmLVV, PVAmLVV, and V̇o2 intercept seem to be causally related to increases of the proteolysis of α-fodrin (Figs. 6 and 7).
Oxygen cost of LV contractility (equals eEmax).
In each eight heart underwent Ca2+ infusion protocol in normal, post-CP, and post-CP + SNJ groups, ESPmLVV and V̇o2 at mLVV (V̇o2mLVV) were measured (Ca2+ ino-run). There were no differences in increases of ESPmLVV and V̇o2mLVV among normal, post-CP, and post-CP + SNJ groups. The linear relations of PVA-independent V̇o2 and eEmax during the Ca2+ infusion in normal, post-CP, and post-CP + SNJ group hearts could be superimposed (data not shown). There were no significant differences in mean oxygen costs of LV contractility among the three groups (Fig. 8). This indicated that V̇o2 used for Ca2+ handling in E-C coupling per unit changes in LV contractility was not different among the three groups.
Immunoblotting of LTCC and SERCA2a and logistic time constants in normal, post-CP, and post-CP + SNJ group hearts.
Figure 9A showed immunoblottings of LTCC and SERCA2a in each group heart. The mean amounts of LTCC and SERCA2a were not significantly different among normal, post-CP, and post-CP + SNJ groups. Figure 9B showed that the mean logistic time constants of one-beat LV pressure-time curve at mLVV were not significantly different among normal, post-CP, and post-CP + SNJ groups.
The most important finding in the present study was that a newly developed water soluble calpain inhibitor SNJ antagonized post-CP LV contractile dysfunction associated with decreased Ca2+ handling V̇o2 in E-C coupling and unchanged basal metabolic O2 consumption without changing the oxygen cost of PVA (oxygen cost of mechanical energy; see Ref. 32) or oxygen cost of LV contractility (oxygen cost of Ca2+ handling energy in E-C coupling per unit change in LV contractility; see Refs. 32, 33, and 38). SNJ also antagonized α-fodrin proteolysis in post-CP without any effects on amounts of LTCC and SERCA2a.
Previous studies have demonstrated that the lipophilic calpain inhibitor calpain inhibitor-1 attenuated α-fodrin proteolysis and cardiac dysfunction due to I/R injury (42) and acute Ca2+ overload (37). In the present study, a water-soluble calpain inhibitor SNJ at 40 μM, which was fourfold higher than the cardioprotective concentration (10 μM) of a lipophilic calpain inhibitor-1 (42), also attenuated α-fodrin proteolysis and attenuated the cardiac dysfunction due to the impairment of total Ca2+ handling in E-C coupling in post-CP group.
It has been proposed that fodrin maintains the integrity of the plasma membranes as a constituent of the membrane skeleton (3, 17). Therefore, it seems likely that the degradation of fodrin in membrane fractions would alter the properties of ion channels (40). The present results showing decreased Ca2+ handling V̇o2 in E-C coupling associated with unchanged oxygen cost of PVA and unchanged oxygen cost of LV contractility probably reflect the decreased total amount of Ca2+ handled, which may be due to a suppression of the transsarcolemmal Ca2+ influx (13, 32, 37, 42). From the possibility that disruption of cytoskeletal proteins inactivates LTCC (8, 22), we speculate that the linkage of the LTCC to the membrane fodrin tethers the channel in place, which somehow modulates the basal activity of the channel, and a loss of the linkage may impair its regulation. Therefore, the calpain inhibitor may have protected against LV dysfunction by preserving the structural integrity of the LTCC in the cell membrane (8, 22).
However, recent studies have revealed that calpain activation induced by I/R injury causes degradation of Ca2+ handling proteins such as LTCC (6) and SERCA2a (7). This was demonstrated by application of a lipophilic calpain inhibitor-3 (10 μM). In the present study, we presumed that these mechanisms also might have contributed to Ca2+ handling dysfunction and that SNJ might have inhibited degradation of SERCA2a (7) and LTCC (6). However, the present study indicates that 30 meq KCl containing cardioplegic cardiac arrest and reperfusion never caused degradation of SERCA2a and LTCC. In addition, LV lusitropism revealed by logistic time constants (21) was not significantly different among normal, post-CP, and post-CP + SNJ groups, indicating that Ca2+ uptake function by SERCA2a was functioning normally.
In conclusion, the present results suggested that a novel water-soluble calpain inhibitor SNJ added in cardioplegia and reperfusate exerted a marked cardioprotective action. To determine the clinical feasibility of SNJ-1945 in cardioplegic cardiac arrest-reperfusion, in vivo studies are clearly needed.
This work was supported in part by Grants-in-Aid for Scientific Research 19591643 and 19790190 from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also supported in part by Biotechnology and Biological Sciences Research Council Japan Partnering Programme 2007-2011 with support from the Biotechnology and Biological Sciences Research Council.
No conflicts of interest are declared by the author(s).
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