SPECT imaging of cytochrome c in pressure overload mice hearts

Abstract

Clinically, pressure overload (PO) occurs in many clinical settings such as hypertension and valvular stenosis especially in the current aging society. During cardiac remodeling induced by PO, mitochondria were injured and cytochrome c was released. However, few studies have targeted cytochrome c for imaging. In this study, Technium-99m labeled cytochrome c antibody was developed to image PO hearts induced by transverse aortic constriction surgery. ^99mTc-Anti-cytochrome c was available to image and differentiate cytochrome c in normal mice hearts and PO hearts. The findings on images were consistent with detection by cardiac function and molecular biological assay. Hence, the noninvasive targeted cytochrome c imaging of mice hearts can be realized via ^99mTc-Anti-cytochrome c based SPECT.

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Introduction

Pressure overload (PO) is a common cardiac stress which imposes increased hemodynamic afterload on the left ventricle (LV). It is encountered in numerous clinical settings such as valve stenosis and hypertension,^1,2 which is one of the most important and growing reasons for cardiovascular diseases in the current aging society. Prominent cardiac remodeling occurred in response to PO, involving a vast array of structural, functional, inflammation, signaling and metabolic alterations.^3,4 Although the remodeling process is complex, accumulating evidence has indicated that mitochondria play a central critical role in PO hearts.^5,6

Current thinking holds that mitochondria are far more referred to as the ‘energy plants’ of the cell, they are involved in the control of survival or death in cardiac myocytes through regulating apoptosis and necrosis.⁷ Cytochrome c is the well known protein released from mitochondria to cytosol and trigger apoptosis⁸ under various cardiac stresses, which underlies the pathogenesis of many cardiovascular diseases. A large number of clinical trials have shown plasma cytochrome c is not only an initiator of apoptosis but also a marker of mitochondrial injury which was tightly associated with the severity of disease and clinical prognosis.^9,10 Thus, detecting cytochrome c may provide a reliable and valuable way to reflect mitochondrial injury and subsequent apoptosis cascade.

By now, ultrasound based evaluation of ventricular morphologic changes was mainly used as noninvasive tool for human PO hearts. However, it only provides limited and hysteretic information. Recent advances of noninvasive molecular imaging have proven great potential in both research and clinical settings. Quantitative positron emission tomography imaging with ¹⁸F-FDG was developed to assess metabolic remodeling in PO patients.¹¹ However, few studies have targeted early cytochrome c changes for imaging. Given that the vital role of cytochrome c in cardiovascular diseases, it is promising to detect cytochrome c alternations exposed to cardiac stress in vivo. Here, we imaged the cytochrome c based on SPECT in transverse aortic constriction induced PO hearts. The noninvasive assessment of cytochrome c may help provide more information about emerging mitochondrial injury and guide clinical management of PO associated patients.

Experimental

Pressure-overload model and echocardiography

This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals, Eighth edition (2011). All procedures were approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University. Twelve-week-old male, C57BL/6J mice were subjected to transverse aortic constriction surgery to induce PO. In brief, mice were anesthetized by isoflurane and ventilated. As described previously,¹² nylon suture was used to tie the aorta around a blunted 27-gauge needle between the innominate artery and left common carotid artery. After needle removal, a defined stenosis of aorta was produced. The control mice underwent the same operation without ligation of the aorta. Echocardiography (VisualSonics, Toronto, Canada) was performed at 3 weeks after TAC surgery under isoflurane mixed with O₂ to provide adequate ventilation and sedation. Left parasternal long-axis view was used to assess left ventricular systolic function and structure, parameters including left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), left ventricular anterior wall end-systolic (LVAWS) and end-diastolic thickness (LVAWD), left ventricular posterior wall end-systolic (LVPWS) and end-diastolic thickness (LVPWD), left ventricular end-systolic (LVESD) and end-diastolic (LVEDD) dimensions were recorded.

Preparation of Technium-99m labeled cytochrome c antibody

Anti-cytochrome c antibody (Abcam, USA) was labeled with freshly eluted ^99mTcO₄⁻ using a reported method with minor modifications.¹³ For one mouse's dose of imaging agent, 10 μg antibody in 10 μL 0.3 M HEPES buffer (pH = 8.0) was mixed with 1 μg NHS-MAG3 (in 1 μL DMSO) synthesized in house.¹⁴ The mixture was vortexed and incubated for 1 h. The reaction system was transferred to a filter tube with 10 kD filtering limit to remove the free NHS-MAG3, and conjugate was dissolved in 0.25 M NH₄OAc. For radiolabeling, 20 μL tartrate buffer (pH = 8) was then added to the above conjugate, and no more than 18.5 MBq ^99mTcO₄⁻ eluent was added. After that, 5 μL freshly prepared SnCl₂·2H₂O (1 mg mL⁻¹, in 0.01 M HCl) solution was added to the mixture and incubated for 30 min at room temperature. The schematic procedure of preparation of imaging agents was shown in Fig. 1A.

Fig. 1 Preparation of imaging agent and quality controls, including labeling rates and in vitro stabilities.

The quality controls, including labeling rate and radiochemical purity of ^99mTc-MAG3-Anti-CyC (abbreviated as ^99mTc-Anti-CyC, herein and after) were determined using radio-thin layer chromatography (TLC) in two systems. The first system was with acetone as developing solvent and silicon plate as stationary phase. The labeled product and collides stayed at the original point (R_f = 0) and the free Technium-99m moved to the top (R_f = 1). The second system was with 0.9% NaCl as developing solvent and whatman 3MM chromatography paper as stationary phase. The labeled product and free Technium-99m moved to the top (R_f = 1) and the collides stayed at the original point (R_f = 0). Labeling rates and radiochemical purity were calculated based on the results from two TLC systems. The in vitro stability of the ^99mTc-Anti-CyC was evaluated in 0.1 M phosphate-buffered saline (PBS) and 10% serum solution (w/v) by radio-TLC with the above two systems. The radiolabeled antibody was diluted with a ratio of 1 : 10 in PBS and incubated for another 6 hours at room temperature. To estimate the stability in serum, the labeled product was mixed with equal volumes of the tracer and 10% serum solution after incubation for 6 h at 37 °C.

SPECT/CT imaging of animal models

^99mTc-Anti-CyC-based SPECT/CT. To detect in vivo mechanism, ^99mTc-Anti-CyC (18.5 MBq per mouse, 10 μg antibody included) was administered to control mice via tail vein injection. Mice were kept for free of food and water. At 1.5 h or 3 h after injection, mice were anesthetized via 2% isoflurane inhalation. CT was performed first with parameters as below: frame resolution, 256 × 512; tube voltage, 45 kVp; tube current, 0.15 mA; exposure time, 500 ms per frame. Real-time 3D reconstruction was performed using the Nucline software (v 1.02, Mediso, Budapest, Hungary). SPECT was performed following the CT scanning with the same scanning scope using the following parameters: four high-resolution, conical collimators with 9-pinhole plates; energy peak, 140 keV; window width, 10%; resolution, 1 mm per pixel; matrix, 256 × 256; scan time, 20 s per projection with 24 projections in all. Three-dimensional ordered-subset expectation maximization images were reconstructed using HiSPECT (Bioscan, Washington, DC). The whole-body SPECT/CT scan for each mouse took 24 min on average.

For further myocardial cytochrome c detection, PO mice were injected with ^99mTc-Anti-CyC and scanned at the proper time point. The regions of interest (ROIs) were manually drawn on the axial sections to outline the heart, and signal ratios of heart to surrounding tissues were measured.

Histology and electron microscopy

Cross sections of the pressure-overload and control mice hearts were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were stained with hematoxylin and eosin and Masson's trichrome.

Freshly excised left ventricular samples were fixed in 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide. Mitochondrial morphology and myofiber ultrastructure were observed by microscope (CM-120, Philips, Amsterdam, Netherlands).

Caspase-3 activity assay

Caspase-3 activity was detected using caspase-3 colorimetric assay kit (Abcam, USA) according to manufacturer's protocol. Briefly, fresh left ventricular tissues were homogenized using homogenizer in chilled lysis buffer. Then, each sample was mixed well with 50 μL of 2× reaction buffer (containing 10 mM DTT) and 5 μL of the 4 mM DEVD-p-NA substrate. After incubating at 37 °C for 90 min, optical density was measured at OD400 nm.

Mitochondrial aconitase activity

Mitochondrial fractions from fresh left ventricular homogenate were resuspended in sodium citrate and aconitase activity was detected using aconitase activity assay kit following the manufacturer's instructions (OxisResearch, USA). In brief, mitochondrial samples were incubated with substrate, enzyme and NADP in a 96-well plate for 15 min at 37 °C. The absorbance was recorded at 340 nm by a spectrophotometer. Tris–HCl buffer was served as blank.

Western blot analysis

Left ventricular tissue was used for western immunoblotting as previously described¹⁵ using antibodies against cytochrome c (Abcam, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control (Kangchen Biotechnology, Nanjing, China). Band intensity of proteins of interest was normalized to that of GAPDH.

Statistical analysis

The data were expressed as mean ± SEM and analyzed using SPSS (v.16.0, SPSS Inc., Chicago, IL, USA). Student's t test was used to compare control and PO mice. P < 0.05 was considered statistically significant.

Results and discussion

Preparation of ^99mTc-Anti-CyC

As shown in Fig. 1B, anti-CyC can be labeled with Technium-99m with labeling rate of 96.2 ± 0.7% with a small quantity of collides existed. The specific activity can be as high as 18.5 MBq per μg without decreasing the labeling rate. Fig. 1C showed the in vitro stability of ^99mTc-Anti-CyC that no obvious de-labeling was observed in PBS buffer, and a slight decrease of radiochemical purity was observed in serum solution. For in vivo application as imaging agent, the specific activity was set as 18.5 MBq/10 μg, and imaging agent was used without further purification and diluted with 0.9% NaCl solution.

^99mTc-Anti-CyC based SPECT/CT

For the in vivo mechanism shown in Fig. 2, imaging tracers were mainly expelled via renal-urinary route. Livers also uptake abundant of ^99mTc-Anti-CyC. More than 20.5% imaging agents were expelled from body during the first 1.5 hours, and more than 45.3% tracers were expelled at 3 hours post injection. SPECT/CT scanning of PO mice was performed at 1.5 hours post injection in consideration that the surrounding tissues of heart were of low tracer uptake and a relative long time was meaningless for getting a clear background. On the other side, short time period can maintain the in vivo radiochemical purity. At 1.5 hours post injection intravenously, no obvious accumulation in thyroid was observed, proving the in vivo stability during the SPECT/CT scanning.

Fig. 2 ^99mTc-Anti-CyC based SPECT/CT imaging of normal mice at 1.5 h and 3 h post injection.

At 1.5 hours post injection, injured myocardium was clearly showed on the axial sections via the SPECT signal from ^99mTc-Anti-CyC uptake. PO cardiomyocytes uptake ^99mTc-Anti-CyC significantly more than surrounding lung tissues (1.16 ± 0.13% ID mL⁻¹ vs. 0.32 ± 0.12% ID mL⁻¹, P < 0.05) and normal cardiomyocytes (1.16 ± 0.13% ID mL⁻¹ vs. 0.21 ± 0.15% ID mL⁻¹, P < 0.05) (Fig. 3).

Fig. 3 ^99mTc-Anti-CyC based SPECT/CT imaging of control (left) and PO (right) mice hearts at 1.5 h post injection. The sagittal, coronal and transversal sections were provided to show the different on anti-cytochrome c targeting. Additionally, three typical transversal sections of hearts were provided and the corresponding positions were labeled with red lines.

Cardiac function

Three weeks after TAC surgery, heart weight was significantly increased in PO mice. Compared to the control mice, echocardiography results showed that cardiac function was impaired in PO mice as evidenced by decreased EF and FS (P < 0.05). Concurrently, wall thicknesses of PO mice including LVAWS, LVPWS were significantly increased (P < 0.05) which exhibited a typical PO remodeling (Table 1).

Table 1 Cardiac function of control and PO mice^a

	Pressure overload remodeling
	Control	PO
a Heart/weight: heart weight to body weight ratio; LVEDD: left ventricular end-diastolic dimensions; LVESD: left ventricular end-systolic dimensions; LVAWS: left ventricular anterior wall end-systolic thickness; LVAWD: left ventricular anterior wall end-diastolic thickness; LVPWS: left ventricular posterior wall end-systolic thickness; LVPWD: left ventricular posterior wall end-diastolic thickness; LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional shortening; *P < 0.05 PO vs. control.
Heart weight (g)	0.15 ± 0.02	0.20 ± 0.01*
Heart/weight	0.42 ± 0.01	0.72 ± 0.03*
LVEDD	3.40 ± 0.06	3.90 ± 0.07*
LVESD	2.30 ± 0.07	2.96 ± 0.08*
LVAWS	0.95 ± 0.03	1.18 ± 0.02*
LVAWD	0.71 ± 0.04	0.92 ± 0.03*
LVPWS	0.91 ± 0.01	1.12 ± 0.03*
LVPWD	0.67 ± 0.02	0.89 ± 0.02*
LVEF	79.38 ± 2.35	45.40 ± 1.14*
LVFS	46.82 ± 1.18	22.62 ± 0.92*

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Histology and mitochondrial ultrastructure

Histology by HE staining and masson's trichrome staining indicated that the volume of cardiomyocytes and interstitial fibrosis were significantly increased in PO mice compared to control mice (Fig. 4A and B). Furthermore, electron microscopy results indicated that the ultrastructure of myocardium especially mitochondria were profoundly affected by PO stress. The mitochondria of PO mice were characterized by damaged morphology phenotype such as mitochondrial swelling, vacuolation and disruption of cristae (Fig. 4C).

Fig. 4 Histology and ultrastructure of LV of control and PO mice. (A) Representative images of hematoxylin and eosin (HE) staining (400×). (B) Representative images of Masson's trichrome staining (200×). (C) Representative TEM images of the LV in control and PO mice. Scale bar, 0.5 μm.

Cytochrome c, early apoptosis and mitochondrial injury markers in PO hearts

The cytochrome c expression in control and PO mice hearts were further detected. As shown in Fig. 5A and B, there was a significantly increased expression of cytochrome c in LV of PO mice than control mice (P < 0.05), which was in good consistent with ^99mTc-Anti-CyC based SPECT/CT imaging results.

Fig. 5 Western blot analysis of cytochrome c and activity assay of caspase-3 and mitochondrial aconitase in control and PO mice. (A) Representative blots of cytochrome c and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (loading control). (B) Cytochrome c expression (n = 4). (C) Caspase-3 activity of LV. (D) Aconitase activity of LV. Values are expressed as mean ± SEM (n = 4–6 per group). *P < 0.05 PO vs. control.

Furthermore, cytochrome c associated apoptosis molecule-caspase 3 activity was detected. Caspase 3 activity was more elevated in PO mice than control mice (P < 0.05) (Fig. 5C). Meanwhile, aconitase was evaluated as mitochondrial injury marker, which is a vital mitochondrial citric acid cycle enzyme and is readily impaired by oxidative stress. Our data showed that aconitase activity was significantly reduced in PO mice compared to control mice, demonstrating that mitochondria were damaged in PO stress (Fig. 5D).

Cytochrome c is normally located in the outer surface of the inner mitochondrial membrane and exerts its key physiological role in oxidative phosphorylation by transferring electrons between complex III and complex IV.^7,16 Far beyond its crucial energy metabolism importance, it is regarded as an initiator of apoptosis which is released from mitochondria to cytoplasm and subsequently activates apoptosis executor-caspase 3. Therefore, released cytochrome c and activated caspase 3 are often considered as an early apoptosis event under various cardiac stresses.^17,18 In our study, cytochrome c and caspase 3 activity were significantly increased in PO mice, which was in good consistent with this notion. Current thinking holds that cytochrome c is a novel and reliable mitochondrial injury marker in patients under many different diseases such as cancer therapy,^19,20 acute myocardial infarction,¹⁰ systemic inflammatory response syndrome²¹ and encephalopathy,^9,22 which highlight its potential importance of clinical values. Indeed, the level of cytochrome c is a precise indicator of the severity of the disease and clinical prognosis of patients. Abnormal mitochondrial morphology revealed from electron microscope combined with decreased mitochondrial aconitase activity not only pointed out that the mitochondria were injured in PO mice, but also underlined the possibility of cytochrome c imaging as a mitochondrial injury marker.

Recent advances in molecular targeted imaging have helped to identify key molecules of interest in biological process especially in apoptosis. Annexin V is the imaging agent which is under clinical investigation,^23,24 although it was proven promising in several clinical trials, it suffered from some drawbacks such as transient nature of phosphatidylserine (PS) exposure and a limited time window.²³ Given cytochrome c is not only a marker of apoptosis but also involved in mitochondrial injury and energy metabolism, our imaging results demonstrated targeted cytochrome c may be a promising way to monitor and evaluate cardiac diseases. It is noteworthy that most noninvasive imaging of apoptosis belongs to the detection of PS on the cell membrane, which attributes to their relative accessibility. A large number of studies have shown that cytochrome c can reach the extracellular space and appears in the bloodstream without concomitant cell necrosis.^20,25,26 Therefore, the visualized cytochrome c in our study may locate in extracellular space on account of cytochrome c release or in intracellular space on account of cell membrane permeability or other transportation pathway changes under PO stress. Although ^99mTc-Anti-CyC based SPECT was proved available to image and differentiate cytochrome c in normal mice hearts and PO hearts, the mechanism needs to be further studied.

Conclusion

In this study, ^99mTc-Anti-CyC based SPECT realized the noninvasive detection of PO induced significant cardiac remodeling and mitochondrial damage. Given the essential role of cytochrome c, visualizing cytochrome c in vivo which provided a promising way to detect and evaluate myocardial diseases, benefiting the prevention and treatment.

Acknowledgements

This work was supported by National Natural Science Foundation of China (81570224).

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