2013 年 36 巻 4 号 p. 529-539
The mechanisms underlying mitochondrial impairment in the failing heart are not yet clearly defined. In the present study, we examined the involvement of changes in small heat shock proteins (HSPs) such as HSPB1, HSPB5 and HSPB8 in mitochondrial dysfunction of the failing heart. Hemodynamic parameters of rats with myocardial infarction at the 2nd and 8th weeks (2W- and 8W-) after coronary artery ligation (CAL) were measured. The 8W-CAL rats, but not the 2W-CAL ones, showed the signs of the chronic heart failure concomitant with a reduced mitochondrial oxygen consumption rate. In the mitochondrial fraction prepared from the heart of the 2W-CAL animals, the contents of small HSPs and phosphorylated small HSPs were increased, suggesting that these increases contributed to the preservation of the mitochondrial energy-producing ability. In the failing heart, HSPB1 and HSPB8 contents and phosphorylated small HSP contents in the mitochondrial fraction were decreased, suggesting that a reduction in mitochondrial translocation of these small HSPs led to impaired mitochondrial energy-producing ability. To further define the submitochondrial locations of these small HSPs, we performed mitochondrial subfractionation. The contents of small HSPs in the 2W-CAL rats were increased in the mitochondrial inner-membrane fraction, whereas those of the 8W-CAL rats were reversed to those of the control animals. These findings suggest that small HSPs, at least in part, play an important role in the development of the impaired mitochondrial energy-producing ability that leads to heart failure after a myocardial infarction.
Human heart failure represents a final common endpoint for heart diseases, leading to cardiovascular death. In previous studies, we showed that decreased levels of myocardial high-energy phosphates in the remaining viable left ventricle were associated with the development of the heart failure following a myocardial infarction (MI).1) However, the mechanisms responsible for mitochondrial dysfunction in the failing heart have not yet been clarified.
As a consequence of an exposure to various forms of stress such as exposure to heat, chemical compounds, and humoral factors, adaptive responses of cells and tissues to this stress occur immediately, which are involved in increased expression of heat shock proteins (HSPs).2) In fact, HSP synthesis transiently rises in the presence of stress stimuli as a mechanism underlying tissue protection against stress-induced tissue damage.3) In mammalian species, there are many HSP subfamilies. The small HSPs are characterized by their molecular size of 12–43 kDa, and they are classified into 2 major groups according to their subcellular localization.4) Particularly, small HSPs such as HSPB1, HSPB5 and HSPB8 are highly expressed in mammalian cardiomyocytes.5,6) It is postulated that these proteins may play an important role in cardiac stress–response induced by ischemia/reperfusion injury and pressure-overload.7–9) In cardiomyocytes, small HSPs act as a molecular chaperone, which may protect the cells from stress-induced metabolic impairments.10) It is known that the phosphorylation of small HSPs plays a pivotal role in the maintenance of cellular functions.11) Upon ischemia/reperfusion, the phosphorylation of small HSPs on their serine residue occurs via the activation of several protein kinases; and this phosphorylation is involved in the tissue-protective ability of small HSPs.11–13) Furthermore, phosphorylated HSPB5 has been suggested to be translocated to the mitochondria in the ischemic/reperfused myocardium.14) Therefore, it has been hypothesized that phosphorylated small HSPs may play key roles in reducing the mitochondrial damage induced by ischemia/reperfusion injury.
In the failing heart following MI, the mitochondrial energy-producing ability of the myocardium in rats is impaired.1) Previous studies of ours showed that the mitochondrial oxygen consumption rate of the failing heart following MI and pulmonary hypertension was decreased, and we found that especially the activity of complex I in the mitochondrial electron transport system was markedly reduced.15) In the present study, to verify the hypothesis that mitochondrial dysfunction in the rat failing heart is associated with alterations in mitochondrial small HSPs, we determined the contents and phosphorylation levels of small HSPs, such as HSPB1, HSPB5 and HSPB8 in the mitochondrial fraction during the development of heart failure in rats after MI.
Male Wistar rats (SLC, Shizuoka, Japan), weighing 220–240 g, were used in the present study. The investigation conformed with the “Guide for the Care and Use of Laboratory Animals” published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). The protocol of the present study was approved by the Committee of Animal Use and Welfare of Tokyo University of Pharmacy and Life Sciences.
Myocardial infarction of rats was produced by ligation of the left ventricular (LV) coronary artery according to the method described previously.1) Rats with myocardial infarction (coronary artery ligation (CAL) rats) with an approximately 40% infarct area in their left ventricle are consistently produced under our experimental conditions.16) Sham-operated rats (Sham rats) were treated in a similar manner except that not coronary artery ligation was performed.
Echocardiographic MeasurementsTransthoracic echocardiography was performed on the CAL (2W-CAL and 8W-CAL) and Sham (2W-Sham and 8W-Sham) rats according to the method described previously.17,18) Rats were anesthetized with 40 mg/kg intraperitoneally (i.p.) pentobarbital sodium, and then their chest hair was shaved off before examination. Two-dimensional and Doppler imaging were performed by using a ProSound 5500® (Aloka, Tokyo, Japan) equipped with a 10-MHz transducer. The transthoracic echocardiographic probe was placed so as to obtain short-axis and long-axis views. The LV internal diameters at end diastole (LVIDd) and systole (LVIDs) were measured, and then the LV fractional shortening (FS) and ejection fraction (EF) were calculated from the LV dimensions. The cardiac output (CO) and stroke volume (SV) at the pulmonary artery and the Tei index19) were measured from the long-axis and apical four-chamber views. After determination of the pulmonary arterial flow, heart rate (HR), velocity time integral (VTI), and pulmonary arterial diameter (PAD) were measured from the long-axis view; and the ratios of CO and SV to body weight (BW) were calculated as CO and SV indices (COI and SVI, respectively).
Invasive Measurement of Hemodynamic ParametersAfter measurement of cardiac functions by echocardiography, hemodynamic parameters of the CAL (2W-CAL and 8W-CAL) and Sham (2W-Sham and 8W-Sham) rats were measured by the cannula method described previously.1,20) Rats were anesthetized with pentobarbital sodium (40 mg/kg i.p.), and the LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), right ventricular (RV) systolic pressure (RVSP), RV end-diastolic pressure (RVEDP), mean arterial pressure (MAP), and heart rate (HR) were measured through a polyethylene catheter connected to a pressure transducer (DX-360, Nihon Kohden, Tokyo, Japan).
Measurement of Mitochondrial Oxygen Consumption in Skinned BundlesThe mitochondrial oxygen consumption rate (OCR) of cardiac tissue was determined by the method described previously.1,20) The isolated hearts were divided into the infarct area and the viable left ventricle. Myocardial fibers, 0.3 to 0.4 mm in diameter and 3 to 4 mm in length, were prepared from the left ventricular free wall by use of a McIlwain Tissue Chopper (Mickle Lab. Engineering Co., NY, U.S.A.) and transferred into medium A of the following composition (mm): ethylene glycol bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 10; MgSO4, 3; taurine, 20; dithiothreitol, 0.5; imidazole, 20; potassium 2-(N-morpholino)-ethanesulfonate, 160; ATP, 5; creatine phosphate, 15 (pH 7.0); KH2PO4, 3. The fibers were incubated for 20 min in 1 mL of medium A containing 75 µg/mL saponin. After incubation, the fibers (skinned fibers) were washed for 10 min in fresh medium B (medium A without ATP and creatine phosphate but supplemented with 0.5% bovine serum albumin) to remove the saponin. All procedures were carried out at 4°C. The oxygen consumption rate of skinned fibers was determined by means of a Clark-type electrode connected to an Oxygraph (Central Kagaku, Tokyo, Japan) containing skinned fibers in 1.0 mL of medium B at 30°C. For determination of the total (maximal) OCR of complex I, II, and cytochrome c/complex IV in the mitochondrial electron-transport system, glutamate/malate, succinate, and ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) were used as the corresponding substrates, respectively. After determination of the oxygen consumption rate, the skinned fibers were solubilized with 0.5 mL of 2 m NaOH for 30 min at 60°C, and then the protein concentration was determined. The mitochondrial oxygen consumption rate was expressed as nano-atoms of oxygen consumed per min per mg protein.
Western Blotting and Detection of ProteinsSample preparation for Western blotting was performed according to the method of Takahashi et al.21) The heart was isolated and divided into LV wall, septum (Sep), and right ventricular (RV) wall; and then these tissues were weighed. Furthermore, the isolated LV wall was separated into viable area (viable LV) and scar tissue and stored at −80°C. The viable LV was homogenized in a homogenization buffer (250 mm sucrose, 20 mm N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 1 mm dithiothreitol (DTT), 1 mm EGTA, cOmplete® protease inhibitor cocktail [Roche, Basel, Switzerland], and PhosSTOP® phosphatase inhibitor cocktail [Roche]; pH 7.4, at 4°C). The homogenate was centrifuged at 1000×g for 10 min, and then the resultant supernatant fluid was centrifuged at 8000×g for 20 min. The pellet was washed twice and suspended in the homogenization buffer, and the suspension was centrifuged at 8000×g for 20 min again, and then the pellet was washed twice and suspended in the homogenization buffer (mitochondrial fraction). The supernatant fluid was centrifuged at 100000×g for 1 h, and then the supernatant fluid was collected (cytosolic fraction). Western blot analysis of small HSPs was performed according to the method described previously with some modifications.21,22) The following antibodies were used: anti-HSPB1 (Enzo Life Science Inc., Farmingdale, NY, U.S.A.), anti-HSPB5 (Enzo Life Science Inc.), anti-HSPB8 (StressMarq, Victoria, Canada), anti-phospho HSPB1 Ser85 (Enzo Life Science Inc.), anti-phospho HSPB5 Ser45 (Enzo Life Science Inc.), anti-phospho HSPB5 Ser59 (Enzo Life Science Inc.), anti-GAPDH (Merck, Darmstadt, Germany), anti-COX IV (Life Technologies Co., Carlsbad, CA, U.S.A.), anti-VDAC (Merck), anti-HSP60 (Enzo Life Science Inc.), anti-MAPKAPK2 (Genscript USA Inc., Piscataway, NJ, U.S.A.), anti-phospho MAPKAPK2 (Genscript USA Inc.), anti-Erk1/2 (Cell Signaling Technology, Inc., Danvers, MA, U.S.A.), and anti-phospho Erk1/2 Thr202/Tyr204 (Cell Signaling Technology, Inc.) antibodies.
Mitochondrial SubfractionationPreparation of inner and outer mitochondrial membrane fractions was performed.23,24) Cardiac mitochondria (50 µL of 10 µg/μL) were re-suspended in 450 µL hypotonic buffer (5 mm Tris–HCl, 1 mm ethylenediaminetetraacetic acid (EDTA) [pH 7.4]) and incubated on ice for 20 min, and then the resultant solution was centrifuged at 20000×g for 10 min at 4°C. The resultant pellet contained the mitoplasts (consisting of the inner membrane [IM] and matrix [MA]). The supernatant was then spun at 120000×g for 60 min. The resultant pellet contained the outer membrane (OM)-enriched fraction, whereas the soluble inter membrane space (IMS)-enriched fraction was the supernatant fluid. Then, the mitoplasts were resuspended in 450 µL of hypotonic buffer and sonicated with a Branson Sonifier® (Emerson, St. Louis, MO, U.S.A.) for 30 s×2 at power level 5 on ice to disrupt the IM. The solution was then spun at 120000×g for 60 min. The resultant pellet contained the IM-enriched fraction, whereas the supernatant fluid was used as the MA-enriched fraction is the supernatant fluid.
StatisticsThe results were expressed as the means±S.E.M. Statistical significance of differences were estimated by using 2-way analysis of variance (ANOVA) followed by Scheffe’s multiple comparisons. Differences with a probability of 5% or less were considered to be significant (p<0.05).
Table 1 shows the changes in the tissue weight of the 2W-CAL and 8W-CAL rats. The LVW/BW ratios of the 2W and 8W-CAL rats were not significantly different from those of the corresponding Sham groups. The SepW/BW ratios of the 2W and 8W-CAL rats were increased to approximately 120% and 175%, respectively, of those of the corresponding Sham rats. Similarly, RVW/BW ratios and LungW/BW ratios of the 2W and 8W-CAL rats were increased as compared with the corresponding ratios for the Sham group values. The RVW/BW ratios of the 2W and 8W-CAL rats were increased to approximately 180% and 290%, respectively, of the corresponding Sham rats. For the LungW/BW ratios, the increases were to approximately 270% and 370%, respectively.
| 2W | 8W | |||
|---|---|---|---|---|
| Sham | CAL | Sham | CAL | |
| BW (g) | 242±3 | 225±2* | 315±3 | 278±4*,# |
| Tissue weight | ||||
| LVW (mg) | 336±10 | 329±14 | 408±9 | 361±6* |
| LVW/BW (mg/g) | 1.39±0.04 | 1.46±0.06 | 1.31±0.03 | 1.30±0.03 |
| SepW (mg) | 134±3 | 151±8 | 148±4 | 228±10*,# |
| SepW/BW (mg/g) | 0.56±0.01 | 0.67±0.03 | 0.47±0.01 | 0.82±0.04*,# |
| RVW (mg) | 129±5 | 211±11* | 134±4 | 344±12*,# |
| RVW/BW (mg/g) | 0.53±0.02 | 0.94±0.05* | 0.43±0.01 | 1.24±0.04*,# |
| HW (mg) | 600±11 | 691±14* | 693±12 | 933±14*,# |
| HW/BW (mg/g) | 2.48±0.04 | 3.07±0.05* | 2.22±0.04 | 3.36±0.07*,# |
| LungW (mg) | 811±12 | 2019±91* | 917±25 | 2967±120*,# |
| LungW/BW (mg/g) | 3.35±0.07 | 8.99±0.46* | 2.91±0.08 | 10.68±0.47*,# |
Each value represents the mean±S.E.M. of 8 experiments. * p<0.05 vs. the corresponding Sham group. # p<0.05 vs. 2W CAL group.
Table 2 shows the cardiac parameters determined by the echocardiographic system at the 2nd and 8th weeks after the surgery. The left ventricular FS, a measure of left ventricular systolic function, of the 2W and 8W-CAL rats was decreased to approximately 38% and 22%, respectively, of that of the corresponding Sham rats. The COI of the 2W-CAL group was not significantly different from that of the 2W-Sham group, whereas this value of the 8W-CAL group was decreased to approximately 72% of the corresponding Sham group. PAAT, an estimate of the pulmonary arterial systolic pressure, was decreased at the 2nd and 8th weeks after the surgery. The LV and RV Tei indexes, increases in which result from the development of LV dysfunction and provide prognostic information on a variety of myocardial conditions, were increased at the 2nd week after the surgery as compared with those values for the corresponding Sham rats; and indexes for of the 8W-CAL group were further increased over those for the 2W-CAL group.
| 2W | 8W | |||
|---|---|---|---|---|
| Sham | CAL | Sham | CAL | |
| M-Mode | ||||
| LVIDd (mm) | 5.60±0.08 | 9.02±0.16* | 5.84±0.14 | 10.06±0.18*,# |
| LVIDs (mm) | 2.82±0.08 | 7.31±0.13* | 2.87±0.14 | 8.99±0.15*,# |
| FS (%) | 49.8±0.8 | 18.9±0.9* | 50.6±1.3 | 10.9±1.0*,# |
| ESV (µL) | 23.0±1.8 | 393.8±20.8* | 25.2±3.4 | 734.6±35.1*,# |
| EDV (µL) | 178±8 | 739±39* | 202±15 | 1026±59*,# |
| EF (%) | 87.2±0.6 | 46.4±1.8* | 87.8±1.0 | 28.1±2.5*,# |
| D-Mode | ||||
| HR (bpm) | 396±2 | 411±8 | 403±4 | 393±9 |
| CO (mL/min) | 103.8±1.3 | 96.6±2.0 | 137.9±2.4 | 82.2±2.2*,# |
| CO index (µL/min/g) | 409±4 | 424±9 | 416±5 | 299±9*,# |
| SV (µL) | 262±4 | 236±9 | 342±7 | 210±9* |
| SV index (µL/g) | 1.03±0.02 | 1.03±0.04 | 1.03±0.01 | 0.76±0.03*,# |
| PAAT (ms) | 29.2±0.4 | 20.1±1.4* | 31.7±0.6 | 16.4±0.6*,# |
| LV E/A ratio | 1.52±0.05 | 1.84±0.05* | 1.55±0.04 | 2.25±0.10*,# |
| LV Tei index | 0.33±0.03 | 0.63±0.05* | 0.34±0.03 | 1.06±0.06*,# |
| RV Tei index | 0.21±0.04 | 0.46±0.04* | 0.20±0.03 | 0.72±0.05*,# |
Each value represents the mean±S.E.M. of 8 experiments. * p<0.05 vs. the corresponding Sham group. # p<0.05 vs. 2W CAL group.
Hemodynamic parameters of the Sham and CAL rats were determined at the 2nd and 8th weeks after the surgery by use of the cannula method (Table 3). The LVEDP was increased at the 2nd week, and then further increased more than 30 mmHg at the 8th week. The RVSP of the 2W-CAL rats was increased to approximately 180% of the corresponding Sham value, and that of the 8W-CAL rats was further increased to 2-fold the Sham value. The heart rate did not change throughout the experiment. There were no changes in these hemodynamic parameters of the Sham rats throughout the experiment, when Sham values were compared with those for naïve rats.
| 2W | 8W | |||
|---|---|---|---|---|
| Sham | CAL | Sham | CAL | |
| LVSP (mmHg) | 142±2 | 134±2* | 145±2 | 127±2* |
| LVEDP (mmHg) | 2.1±0.4 | 22.6±2.1* | 2.0±0.6 | 34.1±2.3*,# |
| LV +dp/dt (mmHg/s) | 10591±473 | 7917±325* | 11349±355 | 6135±376*,# |
| LV −dp/dt (mmHg/s) | −9273±266 | −6080±368* | −9342±628 | −4834±180* |
| RVSP (mmHg) | 31±1 | 56±2* | 32±2 | 61±4*,# |
| RVEDP (mmHg) | 0.75±0.33 | 3.25±0.49* | 1.05±0.41 | 4.00±0.53* |
| RV +dp/dt (mmHg/s) | 2208±51 | 3480±158* | 2215±83 | 3878±32* |
| RV −dp/dt (mmHg/s) | −1513±62 | −2356±202* | −1631±82 | −2491±124* |
| MAP (mmHg) | 111±3 | 106±3 | 114±3 | 102±3* |
| HR (bpm) | 426±8 | 389±9 | 405±11 | 410±20 |
Each value represents the mean±S.E.M. of 8 experiments. * p<0.05 vs. the corresponding Sham group. # p<0.05 vs. 2W CAL group.
Changes in mitochondrial OCR of the skinned bundles prepared from the viable LV of the CAL and Sham rats are shown in Fig. 1 (n=each 6). The OCR value for mitochondrial complex I of the myocardial skinned bundles from the 2W-CAL rat was not significantly different from that for the 2W-Sham rat; whereas for the 8W-CAL rats, this value was decreased to approximately 70% of the corresponding Sham value. In contrast, the OCRs of the LV muscle of the 2W- and 8W-CAL rats in the presence of succinate as a substrate for complex II or ascorbate/TMPD for cytochrome c/complex IV were similar to those for the corresponding Sham rats.
Respiration rates were measured by using glutamate plus malate (for complex I), succinate (for complex II) or ascorbate plus TMPD (N,N,N′,N′,-tetramethyl-p-phenylenediamine, for complex IV). Each value represents the mean±S.E.M. of 6-independent experiments. * p<0.05 vs. the corresponding Sham group. # p<0.05 vs. 2W CAL group.
Figure 2 shows changes in the small HSP contents of the viable LV from CAL rats and the LV from Sham rats at the 2nd and 8th week after the operation. The myocardial and cytosolic HSPB1 contents of the 2W-CAL rat were increased to approximately 140% of those for the corresponding Sham rat; whereas those contents of the 8W-CAL rat were similar to those for the corresponding Sham rat (Figs. 2B, E). The myocardial HSPB5 contents of the 2W- and 8W-CAL rats were similar to those for the corresponding Sham rats (Fig. 2C). The HSPB5 content in the cytosolic fraction prepared from the viable LV of the 2W-CAL rat was increased to approximately 120% of that for the corresponding Sham rat, whereas that content of the 8W-CAL rat was similar to that of the corresponding Sham rat (Fig. 2F). As for the myocardial HSPB8 content in the 2W-CAL rats it was increased to approximately 120% of that for the corresponding Sham rats, however, the value for the 8W-CAL rats was decreased to approximately 70% of the 8W-Sham value (Fig. 2D). The HSPB8 contents in the cytosolic fraction prepared from the viable LV of the 2W- and 8W-CAL rats were similar to those for the corresponding Sham rats (Fig. 2G).
Total homogenates, mitochondrial fraction, and cytosolic fraction of the (viable) left ventricle from Sham rats and CAL rats at the 2nd and 8th weeks after surgery were analyzed by immunoblotting. Bands corresponding to HSPB1, HSPB5, and HSPB8 were scanned, and the scanned bands were normalized by GAPDH, COXIV or CBB staining on the same blot. Representative quantified data for HSPB1, HSPB5, and HSPB8 protein contents are shown in the lower panels. Each value represents the mean±S.E.M. of 6-independent experiments. * p<0.05 vs. the corresponding Sham group. # p<0.05 vs. 2W CAL group.
In the mitochondrial fraction, the HSPB1 content prepared from the viable LV of the 2W-CAL rat was increased to approximately 125% of that for the corresponding Sham rat; but this content in the 8W-CAL rat was decreased to approximately 55% of the 8W-Sham value (Fig. 2H). The HSPB5 content of the 2W-CAL and 8W-CAL rats was increased to approximately 130% and 230%, respectively, of that for the corresponding Sham rats (Fig. 2I). The HSPB8 content of the 2W-CAL rat was increased to approximately 170% of that for the corresponding Sham rat; but, in contrast, the mitochondrial HSPB8 content of the 8W-CAL rat was decreased to approximately 50% of the 8W-Sham value (Fig. 2J).
Phosphorylated-Small HSPs Contents in the Viable LVFigure 3 shows changes in the contents of phosphorylated small HSPs in the viable LV from CAL rats and the LV from Sham rats at the 2nd and 8th week after the operation. In the heart, the p-HSPB1 Ser85 content of the 2W- and 8W-CAL rats was increased to approximately 610% and 440%, respectively, of that of the corresponding Sham rats (Fig. 3B). The p-HSPB5 Ser45 content of the 2W- and 8W-CAL rats was increased to approximately 160% and 220%, respectively, of that of the corresponding Sham rats (Fig. 3C); whereas the p-HSPB5 Ser59 contents of both groups were similar to those of the corresponding Sham rats (Fig. 3D).
Total homogenates, mitochondrial fraction, and cytosolic fraction of the (viable) left ventricle from Sham rats and CAL rats at the 2nd and 8th weeks after surgery were analyzed by immunoblotting. Bands corresponding to phosphorylated HSPB1 and HSPB5 were scanned, and the scanned bands were normalized by GAPDH, COXIV or CBB staining on the same blot. Representative quantified data for phosphorylated HSPB1 and HSPB5 protein contents are shown in the lower panels. Each value represents the mean±S.E.M. of 6-independent experiments. * p<0.05 vs. the corresponding Sham group. # p<0.05 vs. 2W CAL group.
In the cytosolic fraction, the p-HSPB1 Ser85 content of the 2W- and 8W-CAL rats rose to approximately 400% and 540%, respectively, of that of the corresponding Sham rats (Fig. 3E). For the p-HSPB5 Ser45 content of the 2W- and 8W-CAL rats, it was elevated to approximately 180% and 370%, respectively, of that of the corresponding Sham rats (Fig. 3F). The p-HSPB5 Ser59 content of the 2W-CAL rat was increased to approximately 150% of that of the corresponding Sham rat, whereas that of the 8W-CAL rat was similar to that of the corresponding Sham rat (Fig. 3G).
In the mitochondrial fraction, the p-HSPB1 Ser85 content of the 2W-CAL rat was increased to approximately 140% of that of the 2W-Sham rat, whereas that of the 8W-CAL rat was decreased to approximately 55% of the 8W-Sham value (Fig. 3H). In the case of the p-HSPB5 Ser45 content of the 2W-CAL rats, it rose to approximately 520% of that of the 2W-Sham rat, whereas 8W-CAL rat and corresponding Sham rat contents were similar (Fig. 3I). The p-HSPB5 Ser59 content of the 2W-CAL rat was increased to approximately 520% of that of the 2W-Sham rat, whereas that of the 8W-CAL rat was decreased to approximately 50% of this content for the corresponding Sham rat (Fig. 3J).
Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MAPKAPK2) and Extracellular Signal-Regulated Kinase 1/2 (Erk1/2) Contents in the Viable LVChanges in MAPKAPK2 and Erk1/2 contents of the viable LV from CAL rats and the LV from Sham rats at the 2nd and 8th week after the operation are shown in Fig. 4. Myocardial MAPKAPK2, Erk1, and Erk2 contents of the 2W- and 8W-CAL rats were larger than those of the corresponding Sham rats (Figs. 4B, E, H). The contents of myocardial MAPKAPK2 phosphorylated at its Thr334 of the 2W- and 8W-CAL rats were increased to approximately 170 and 140%, respectively, of those of the corresponding Sham rats (Fig. 4C). For Erk1 phosphorylated at its Thr202, its content in the 2W- and 8W-CAL rats was increased to approximately 260 and 600%, respectively, of that of the corresponding Sham rats (Fig. 4F). In the case of Erk2 phosphorylated at its Tyr204, its contents in the 2W- and 8W-CAL rats were elevated to approximately 140 and 280%, respectively, of those of the corresponding Sham rats (Fig. 4I).
Total homogenates of the (viable) left ventricle from Sham rats and CAL rats at the 2nd and 8th weeks after surgery were analyzed by immunoblotting (A). Bands corresponding to MAPKAPK2 and Erk1/2 were scanned, and the scanned bands were normalized by GAPDH, COXIV or CBB staining on the same blot. Representative quantified data for MAPKAPK2 and Erk1/2 protein contents are shown in the lower panels (B–J). Each value represents the mean±S.E.M. of 6-independent experiments. * p<0.05 vs. the corresponding Sham group. # p<0.05 vs. 2W CAL group.
We performed a mitochondrial subfractionation study using mitochondria isolated from the viable LV from CAL rats and the LV from Sham rats at the 2nd and 8th week after the operation. Fractions of the outer-membrane (OM), the inter-membrane space (IMS), inner-membrane (IM), and matrix (MA) were prepared. All fractions were then tested by immunoblotting for VDAC as a marker for the OM fraction, COX IV for IM fraction, and HSP60 for the MA fraction.
As shown in Fig. 5A, in control rats HSPB1 was detected in the MA fraction, but found in a lesser amount in the IM fraction. HSPB5 was detected in the OM fraction, with a lower amount in IM and MA fractions; and HSPB8 was detected mainly in the OM fraction, but a lesser amount of it was observed in the IM fraction.
The mitochondrial outer-membrane (OM), inter-membrane space (IMS), inner-membrane (IM), and matrix (MA) fractions of the left ventricle from the control rat were analyzed by immunoblotting (A). The OM, IM, and MA fractions of the (viable) left ventricle from Sham rats and CAL rats at the 2nd and 8th weeks after surgery were analyzed by immunoblotting (B). Bands corresponding to HSPB1, HSPB5, and HSPB8 were scanned and representative quantified data for HSPB1, HSPB5 and HSPB8 protein contents are shown (C–E). Each value represents the mean±S.E.M. of 6-independent experiments. * p<0.05 vs. the corresponding Sham group. # p<0.05 vs. 2W CAL group.
The results of the subfractionation study are shown in Figs. 5B–E. In the OM fraction, compared with that for the Sham rat the content of HSPB1 in the 2W-CAL rat was increased; but in the failing heart at 8 weeks it dropped below the value for the 2W-CAL rat. The content of HSPB8 in the failing heart was decreased to below that in the corresponding Sham rat. In contrast, the HSPB5 content in the OM fraction of the 8W-CAL rat was increased above that of the 8W-Sham rat.
In the IM fraction, the contents of HSPB1, HSPB5, and HSPB8 were increased in the 2W-CAL rats compared with those of the corresponding Sham rats. However, the contents of these small HSPs in the 8W-CAL rats were lower than those of the 2W-CAL rats.
In the MA fraction, the HSPB1 content of the 2W-CAL rat was increased, whereas that of the 8W-CAL rat was decreased, compared with that of the corresponding Sham rats. The HSPB5 content of the 2W-CAL rat was lower than that of the 2W-Sham rat. In contrast, it was increased in the failing heart.
Mitochondrial Localization of Phosphorylated Small HSPs in the Viable LVChanges in the contents of phosphorylated small HSP in the OM, IM, and MA fractions of the viable LV from CAL rats and the LV from Sham rats at the 2nd and 8th week after the operation are shown in Fig. 6.
The mitochondrial outer-membrane (OM), inter-membrane space (IMS), inner-membrane (IM), and matrix (MA) fractions of the (viable) left ventricle from Sham rats and CAL rats at the 2nd and 8th weeks after surgery were analyzed by immunoblotting (A). Bands corresponding to Ser85-phosphorylated HSPB1, and Ser45- and Ser59-phosphorylated HSPB5s were scanned and representative quantified data for HSPB1, HSPB5 and HSPB8 protein contents are shown (B–D). Each value represents the mean±S.E.M. of 6-independent experiments. * p<0.05 vs. the corresponding Sham group. # p<0.05 vs. 2W CAL group.
In both OM and IM fractions, the contents of p-HSPB5 Ser45 and p-HSPB5 Ser59 in the 2W-CAL rat were increased over the Sham values. In the failing heart (8W-CAL), the contents of p-HSPB5 Ser45 and p-HSPB5 Ser59 were decreased compared with those of the 2W-CAL rat.
In the MA fraction, the HSPB1 content of the 2W-CAL rat tended to increase; however, that of the 8W-CAL rat was lower than the content for the 2W-CAL rats. The p-HSPB5 Ser45 contents of both CAL groups were decreased compared with the Sham values, and the p-HSPB5 Ser59 content in the failing heart was less than that in the corresponding Sham rats.
In the present study, we observed a diastolic dysfunction, as suggested by increases in the E/A ratio and LVEDP at the 2nd week after the surgery. Furthermore, decreases in the FS, EF, LVSP, and LV+dp/dt values showed a systolic dysfunction in the 2W-CAL rat. The cardiac pump function, such as indicated by COI and SVI, was preserved in the 2W-CAL rats; whereas decreases in diastolic and systolic functions were observed. These findings suggest that the cardiac function of the 2W-CAL rat was compensated under present experimental conditions. In the 8W-CAL rat, both diastolic and systolic functions were further decreased to levels below those of the 2W-CAL rat; and the COI and SVI were decreased at the 8th, but not the 2nd, week after the surgery. These findings indicate possible signs of chronic heart failure in this model, and are consistent with those in our previous studies.18)
To explore the mitochondrial energy-producing ability, we measured the mitochondrial OCRs of the skinned bundles. The mitochondrial OCR of the viable LV with glutamate as the substrate for complex I in the 2W-CAL rat was preserved. In contrast, the cardiac pump function of the 8W-CAL rats was further decreased, and the animals showed several signs of chronic heart failure associated with the significant decrease in the mitochondrial complex I OCR. These findings suggest that the decline in the mitochondrial OCR of the 8W-, but not 2W-, CAL rats resulted in their inability to produce ATP for preservation of the cardiac pump function.
HSPB1, HSPB5, and HSPB8 are stress-inducible small HSPs that are highly expressed in cardiac tissue.5,6) First, we determined changes in their contents in the cardiac total homogenate, mitochondrial, and cytosolic fractions. The HSPB1 content was significantly increased, and there was a tendency for the HSPB5 and HSPB8 contents to become elevated in the 2W-CAL rats. In the 8W-CAL rat, HSPB1 and HSPB5 contents were similar to those in the 8W-Sham rats; and the HSPB8 content was decreased, suggesting that the ability to induce these small HSPs was attenuated in the failing heart. Such a reduced ability may have played, at least in part, an important role in the genesis of cardiac dysfunction following myocardial infarction.
In the mitochondrial fraction prepared from the heart of the 2W-CAL animals, these three small HSPs were increased in content, suggesting that the mitochondria had been exposed to stress at the compensatory stage of cardiac pump function. Increased levels of these small HSPs in the 2W-CAL rats may have contributed to the preservation of the mitochondrial energy-producing ability. In contrast, HSPB1 and HSPB8 contents were decreased in the mitochondrial fraction from the failing heart. These findings suggest that reduction in these small HSPs led to impaired mitochondrial energy-producing ability.
It is generally known that phosphorylation of proteins regulates their function.25) Next, since we postulated that the phosphorylation of these small HSPs may play a protective role against the progression of heart failure, we determined the contents of phosphorylated small HSPs, such as HSPB1 phosphorylated at its Ser85 and HSPB5, at its Ser45 and Ser59. We found that the amounts of Ser85-phosphorylated HSPB1 and Ser45-phosphorylated HSPB5 were increased in the 2W- and 8W-CAL rat hearts. In the mitochondrial fraction from the 2W-CAL rat, phosphorylated HSPB1 at Ser85 and phosphorylated HSPB5 at Ser45 and Ser59 were increased. In contrast, these contents of the 8W-CAL rats were decreased. The decreases in these phosphorylated small HSPs in the mitochondrial fraction of the failing heart suggest reduced activation of small HSPs. Then, to elucidate the mechanisms underlying this decrease in the levels of these phosphorylatead HSPs, we measured the contents of MAPKAPK2 and Erk1/2, which are protein kinases for HSPB1 and HSPB5. It is now known that HSPB1 is phosphorylated on its serine-85 residue, a phosphorylation site, by protein kinases including MAPKAPK226,27) and that HSPB5 is phosphorylated by MAPKAPK2 (at serine-59) and by Erk (at serine-45).28,29) Contents of active kinases of the 2W- and 8W-CAL rats were increased or tended to be increased compared with those of the corresponding Sham rats. These results are consistent with the increased amounts of phosphorylated small HSPs in the cytosolic fraction of the 2W- and 8W-CAL rats in this study.
To further define the submitochondrial locations of the small HSPs, we prepared mitochondrial OM, IMS, IM, and MA fractions. In the control rat, HSPB5 was mainly detected in the OM fraction, but a small amount of it was found in the IM fraction. HSPB1 was detected in the MA fraction, and HSPB8 was located in the OM one; whereas lesser amounts of these small HSPs were detected in the IM fraction. In the 2W-CAL rat, HSPB1, HSPB5, and HSPB8 were detected in the IM fraction; and the contents of small HSPs in the IM fraction were increased compared with their amounts in the 2W-Sham rat. These findings suggest the possible involvement of the small HSPs in the preservation of the mitochondrial IM-proteins, such as electron-transport enzymes, under the stress condition. In the failing heart, the contents of HSPB1 and Ser85-phosphorylated HSPB1 in the MA fraction, and the contents of Ser45- and Ser59-phosphorylated HSPB5s in the OM fraction, were lower than those of the 2W-CAL rats. Also, the content of HSPB8 in the OM fraction from 8W-CAL rats was decreased compared with that for the 8W-Sham rats. These findings suggest that decreases in the amounts of small HSPs in the mitochondrial fraction, where they are located most abundantly, led to reduced mitochondrial energy-producing ability in the 8W-CAL rat.
In this study, we found HSPB1 including its phosphorylated form and HSPB8 contents were decreased in the mitochondrial fraction from the viable LV of the failing heart. HSPB1 and HSPB8 contents in the viable LV homogenate of the 8W-CAL rats were also decreased compared with those of the 2W-CAL animals. These results show that decreases in HSPB1 and HSPB8 contents in the mitochondrial fraction during the development of heart failure following myocardial infarction are concomitant with those in the homogenate. That is, decreases in mitochondrial HSPB1 and HSPB8 contents may be due to decreases in these HSP contents in the whole heart accompanying a reduction in their synthesis and/or an enhancement of their degradation. Since we also found that the localization of HSPB1 in the submitochondrial fractions was not similar to that of HSPB8, mechanisms of HSPB1 underlying an induction of mitochondrial dysfunction may be different from these pathways of HSPB8 during the development of heart failure. Furthermore, we also found an increase in HSPB5 content and decreases in p-HSPB5 Ser45 and p-HSPB5 Ser59 contents in the mitochondrial fraction from the failing heart. Active forms of protein kinases such as MAPKAPK2 and Erk1/2 in the failing heart were larger than those of the normal heart. In contrast, phosphorylation levels of p-HSPB5 Ser45 and p-HSPB5 Ser59 in the mitochondrial fraction from the failing heart were lower than those in the cytosolic fraction, respectively. These findings suggest that HSPB5 was dephosphorylated by some protein phosphatases in mitochondria, leading to an accumulation of dephosphorylated HSPB5 in submitochondrial fractions such as OM and MA of the failing heart. It is known that there are some protein phosphatases in mitochondria.30,31) Small HSPs are dephosphorylated by the protein phosphatase 2A32) and 2B.33) Therefore, HSPB5 without phosphorylation may be accumulated by these protein phosphatases under the present experimental conditions. Further experiments are required to elucidate the mechanisms underlying changes in small HSPs contents and their phosphorylation levels in the mitochondria during the development of heart failure.
In conclusion, we examined the changes in the small HSPs in the mitochondrial fraction from the rat failing heart following myocardial infarction. Our present results show that the contents of small HSPs and their phosphorylated forms were increased in the 2W-CAL rat, whereas those of HSPB1 and HSPB8, and of phosphorylated HSPB1 and HSPB5, in the mitochondrial fraction from the failing heart were reversed to the levels found for the 8W-Sham rats. Furthermore, when we performed mitochondrial subfractionation, we found that the levels of these small HSPs in the OM and IM fractions were decreased in the failing heart, except for HSPB5 in the OM fraction. These results at least in part suggest that the mitochondrial dysfunction of the failing heart developed via the changes in these small HSPs.
