Author + information
- Received April 3, 2017
- Revision received June 6, 2017
- Accepted July 17, 2017
- Published online September 11, 2017.
- Takashi Tanimoto, MD, PhDa,b,
- Missag H. Parseghian, PhDc,
- Takehiro Nakahara, MD, PhDa,
- Hideki Kawai, MD, PhDa,
- Navneet Narula, MD, PhDd,
- Dongbin Kim, MD, PhDa,
- Robert Nishimura, MDe,f,
- Richard H. Weisbart, MDe,f,
- Grace Chan, BSe,
- Richard A. Richieri, MSc,
- Nezam Haider, PhDa,
- Farhan Chaudhry, MSa,
- Glenn T. Reynolds, MDc,
- John Billimek, PhDg,
- Francis G. Blankenberg, MD, PhDh,
- Partho P. Sengupta, MD, PhDa,
- Artiom D. Petrov, PhDa,
- Takashi Akasaka, MD, PhDb,
- H. William Strauss, MDa and
- Jagat Narula, MD, PhDa,∗ ()
- aIcahn School of Medicine at Mount Sinai, New York, New York
- bWakayama Medical University, Wakayama, Japan
- cRubicon Biotechnology, Lake Forest, California
- dWeill Cornell Medical College, New York, New York
- eVeterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California
- fUniversity of California Geffen School of Medicine, Los Angeles, California
- gUniversity of California, Irvine, Irvine, California
- hLucile Salter Packard Children’s Hospital, Stanford, California
- ↵∗Address for correspondence:
Dr. Jagat Narula, Icahn School of Medicine at Mount Sinai, One Guggenheim Pavilion, 1190 Fifth Avenue, Mount Sinai Heart, N-126, Box 1030, New York, New York 10029.
Background Although early reperfusion is the most desirable intervention after ischemic myocardial insult, it may add to damage through oxidative stress.
Objectives This study investigated the cardioprotective effects of a single intravenous dose of heat shock protein-72 (HSP72) coupled to a single-chain variable fragment (Fv) of monoclonal antibody 3E10 (3E10Fv) in a rabbit ischemia-reperfusion model. The Fv facilitates rapid transport of HSP72 into cells, even with intact membranes.
Methods A left coronary artery occlusion (40 min) reperfusion (3 h) model was used in 31 rabbits. Of these, 12 rabbits received the fusion protein (Fv-HSP72) intravenously. The remaining 19 control rabbits received a molar equivalent of 3E10Fv alone (n = 6), HSP72 alone (n = 6), or phosphate-buffered saline (n = 7). Serial echocardiographic examinations were performed to assess left ventricular function before and after reperfusion. Micro–single-photon emission computed tomography imaging of 99mTc-labeled annexin-V was performed with micro–computed tomography scanning to characterize apoptotic damage in vivo, followed by gamma counting of the excised myocardial specimens to quantify cell death. Histopathological characterization of the myocardial tissue and sequential cardiac troponin I measurements were also undertaken.
Results Myocardial annexin-V uptake was 43% lower in the area at risk (p = 0.0003) in Fv-HSP72–treated rabbits compared with control animals receiving HSP72 or 3E10Fv alone. During reperfusion, troponin I release was 42% lower and the echocardiographic left ventricular ejection fraction 27% higher in the Fv-HSP72–treated group compared with control animals. Histopathological analyses confirmed penetration of 3E10Fv-containing molecules into cardiomyocytes in vivo, and treatment with Fv-HSP72 showed fewer apoptotic nuclei compared with control rabbits.
Conclusions Single-dose administration of Fv-HSP72 fusion protein at the time of reperfusion reduced myocardial apoptosis by almost one-half and improved left ventricular functional recovery after myocardial ischemia-reperfusion injury in rabbits. It might have potential to serve as an adjunct to early reperfusion in the management of myocardial infarction.
The standard of care for acute myocardial infarction is timely reperfusion, usually by percutaneous coronary intervention or thrombolytic therapy. During coronary occlusion, ischemic myocytes endure hypoxic stress and further injury by oxidative stress during reperfusion. The ischemic and oxidative stresses result in irreversible myocardial damage. Pharmacological interventions have been proposed to reduce ischemia-reperfusion injury but have not shown significant efficacy in multicenter clinical trials (1–4). The use of targeted biologic agents that facilitate rapid entry of the therapeutic agent into myocytes might offer enhanced efficacy of reperfusion therapy.
Heat shock proteins (HSPs) are a family of protective chaperone proteins that ensure correct folding and stabilization of newly synthesized critical intracellular proteins or corrective refolding of proteins damaged by oxidative and other cellular stressors. The cardioprotective effects of endogenously produced HSP72 have been previously demonstrated in both in vitro and in vivo studies (5–9). However, >3 h can elapse between endogenous HSP induction by stress stimuli (or even gene therapy) and production of a sufficient amount of intracellular protein for cardioprotection in vivo (10). Messenger ribonucleic acid transcription of HSP70 family members peaks between 2 and 4 h, with significant protein accumulation at 24 h (7). This delay reduces the potential efficacy of strategies that promote endogenous levels of HSP72, such as in the setting of acute myocardial infarction, wherein prompt early intervention is essential.
We propose that carrier-mediated, direct intracellular delivery of HSP72 may facilitate effective cardioprotection in the immediate aftermath of acute myocardial ischemic injury (11). A single-chain variable fragment (Fv) of the monoclonal antibody 3E10 binds deoxyribonucleic acid (DNA) and penetrates viable cells through the energy-independent channel equilibrative nucleoside transporter 2 (ENT2) located in the plasma membrane. To enter cells, 3E10 must bind nucleosides from DNA (12), hence its specificity for tissues undergoing damage where extracellular DNA is accessible. Fusion of the 3E10-Fv to human HSP72 (referred to as Fv-Hsp70 in earlier publications) has been tested in a rat ischemic brain model (13). Recombinant Fv-HSP72 protein demonstrated a more than two-thirds reduction in cell death and provided neuroprotection to cortical motor function.
The goal of the present study was to determine if Fv-HSP72 administration could preserve ischemic myocardium in a rabbit model of left coronary artery ischemia-reperfusion injury. Radiolabeled annexin-V (an in vivo imaging marker of apoptosis) (14), cardiac troponin I levels, and serial echocardiographic assessment of left ventricular ejection fraction (LVEF) were used as objective indicators of myocardial damage.
In a rabbit ischemia-reperfusion model, the cardioprotective effect of the Fv-HSP72 fusion protein was compared versus 3E10-Fv alone or HSP72 alone. The sample of 31 rabbits (12 in the experimental group and 19 control animals) was determined to be sufficient to detect an effect size of Cohen’s d = 1.25 (a group difference in means equivalent to 1.25 SDs for the outcome variable) assuming 80% power and α = 0.017 (applying the Bonferroni correction to account for 3 simultaneous comparisons and a family-wise error rate of 0.05). Animals were randomly assigned to groups. All animals were included in the analysis. Figure 1 summarizes the series of tests and samples collected from each rabbit in the study.
Single-chain Fv was derived from monoclonal antibody 3E10. An Fv-HSP72 fusion protein was designed with the 3E10-Fv located at the N-terminal of the HSP72 protein. Complementary DNA for the Fv was ligated into the plasmid pPICZαA as previously described (15).
Recombinant human annexin-V was obtained from a stock of wild-type annexin-V maintained at −80°C, produced by recombinant techniques under good laboratory practice conditions, as previously described. Annexin-V is a physiological protein that binds avidly to phosphatidylserine expressed on the outer leaflet of the cell membrane in apoptotic cells. Recombinant human–annexin-V was derivatized with the nicotinic acid analogue NHS-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylate hydrochloride) at a 5.0:1.0 mol HYNIC-protein ratio which, after gentle mixing for 3 h at room temperature, resulted in a 0.9:1.0 mol HYNIC-protein product buffer using a previously described protocol (16). HYNIC is a bifunctional chelator with 1 moiety covalently binding the amino group on a lysine side chain on annexin-V and the other moiety conjugating to reduced 99mTc using tricine as a coligand, thereby producing a stable radiocomplex for molecular imaging.
To bind 99mTc to the HYNIC–annexin-V conjugate, 0.2 ml of a reduced tin (stannous ion)/tricine solution was added to a mixture of 20 to 30 mCi of 99mTc pertechnetate and 200 μg of HYNIC–annexin-V under anoxic conditions (16). Radiolabeling efficiency of 99mTc and annexin-V was consistently >90% with a specific activity of 150 to 200 μCi/μg protein.
In vitro studies on human cardiomyocytes
Human primary cardiomyocytes (Catalogue #6200, ScienCell, Carlsbad, California) were thawed and 1 ml (1 × 106 cells/vial) suspended in 19 ml of phenol-red free cardiac myocyte media (includes 5% fetal bovine serum, 1% cardiac myocyte growth supplement, 1X penicillin/streptomycin; Catalogue #6201-prf, ScienCell) before seeding into white, opaque bottom 96-well tissue culture plates at 5,000 cells per well (Catalogue #3917, Corning Life Sciences, Tewksbury, Massachusetts). Cells were incubated overnight at 5% carbon dioxide and 37°C before replacing the media in each well and further incubating for another day.
On the day of experiment, media was replaced with phenol-red free cardiac myocyte media containing 1X celltox green dye (Catalogue #G8741, Promega, Madison, Wisconsin). At time T = 0 h, media with or without hydrogen peroxide (H2O2) was added to each well and incubated for 30 min. The media in each well was further supplemented with 1 of the treatments in Figure 2 and cell death monitored on a 5-mode multidetection plate reader with fluorescence excitation at 490 nm, emission monitored at 525 nm, and a cutoff of 515 nm. Cells were kept at 5% carbon dioxide and 37°C between readings. The percentage of cell death was determined by dividing the average fluorescent signal from each treatment with the average signal obtained after total lysis of cells not intoxicated with H2O2. Statistical significance in Figure 2 was calculated via the Student t test.
Induction of ischemia and reperfusion
Acute experimental myocardial ischemia (17) was produced in anesthetized New Zealand white male rabbits (weight 2.5 to 3.0 kg). The heart was exposed through a parasternal thoracotomy, and the pericardium was removed. A monofilament suture was placed on the lateral branch of the left coronary artery, which was occluded by tightening the snare created by passing the suture through polyethylene tubing and clamping the tube to prevent release of the snare. This protocol follows the Guidelines for the Care and Use of Laboratory Animals established by the National Institutes of Health and approved by the Institutional Animal Care and Use Committee at the Mount Sinai School of Medicine.
At 40 min after occlusion, the snare was released for reperfusion. Rabbits received 1 of 5 treatments, 4 of which were administered via ear vein 1 min before reperfusion. The 4 treatments were: 1) 20 mg (174 nmoles) of 3E10-Fv-HSP72 (Fv-HSP72 pre-reperfusion group; n = 6); 2) an equimolar amount of 3E10-Fv (174 nmol; 3E10-Fv alone group; n = 6); 3) an equimolar amount of HSP72 (174 nmol, HSP72 alone group; n = 6); or 4) phosphate-buffered saline (control group; n = 7). Furthermore, to determine whether there was any significant advantage of Fv-HSP72 administration before reperfusion, we undertook 1 more study group of 20 mg (174 nmoles) of 3E10Fv-HSP72 injected at 1 min after reperfusion (Fv-HSP72 post-reperfusion group, n = 6). Investigators were blinded to the treatment.
To detect apoptosis, ∼7 mCi of 99mTc-labeled annexin-V was injected intravenously 30 min after the start of reperfusion. Rabbits were continuously monitored for another 2.5 h before micro–single-photon emission computed tomography/micro–computed tomography (μSPECT/μCT) imaging. Rabbits were imaged by using a dual-head μSPECT gamma camera combined with μCT scanning.
After in vivo imaging, the left coronary artery was reoccluded at the original site of the snare. Evans blue dye was infused to document the area at risk (AAR) and the region outside the vascular bed of the occluded left coronary artery (the “remote” area) of the heart. The rabbits were killed with an anesthetic overdose of pentobarbital administered intravenously. This method was consistent with the recommendations of the American Veterinary Medical Association Guidelines on Euthanasia. The heart was removed for an ex vivo 1,800 s planar image of the myocardial distribution of 99mTc-annexin-V.
After the ex vivo planar scan, the heart was cut into 4 cross-sectional bread-loaf slices from apex to base (Figure 1). These 4 slices were further sectioned radially into 29 to 33 fragments, which were weighed and counted in a gamma well counter. The percent injected dose (%ID) of 99mTc-annexin-V per gram of tissue was calculated in all myocardial fragments. A mean value of %ID per gram (%ID/g) was calculated in 3 territories; apex (the most vulnerable left ventricular [LV] site in our model), AAR, and remote areas as published previously (17). The total weight of myocardial pieces belonging to AAR, or to the remote areas in each group, did not demonstrate any significant differences, indicating that the sizes of at-risk and unaffected areas were equal in all animals throughout the study.
Cardiac troponin I was measured by using a commercially available enzyme-linked immunosorbent assay kit per manufacturer’s recommendations. Blood samples were collected immediately before coronary occlusion, during occlusion (just before reperfusion), and after reperfusion at 30 min, 1 h, and 3 h. Fresh serum was separated from red blood cells of each blood sample by centrifugation and stored frozen at –80°C for subsequent analysis.
Intraoperative echocardiography was performed by 2 experienced operators using a commercially available cardiac ultrasound equipped with a 5- to 13-MHz linear probe. The ultrasound imaging was performed open chest by directly placing the probe on the heart.
To assess the sequential change of LV global systolic function during the protocol, echocardiography was performed at baseline, 30 min after occlusion, and 3 h after reperfusion per rabbit. In vivo echocardiograms were recorded in the Fv-HSP72 post-reperfusion, 3E10-Fv alone, and the HSP72 alone groups. LV 4- and 2-chamber views were recorded to calculate LVEF by using the modified Simpson’s method (18).
Myocardial tissue specimens from the infarct zone, AAR, and the remote area were obtained for histological characterization. After paraformaldehyde fixation, the specimens were routinely processed and paraffin embedded. Subsequently, 5-μm sections were prepared and stained with hematoxylin and eosin and Masson’s trichrome. Tissue sections were also subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to detect DNA fragmentation during apoptosis per manufacturer’s recommendations. TUNEL-positive cells were counted in 5 high-power fields (400×) showing the maximum number of positive nuclei per section. Finally, sections were immunostained with an antibody for the c-myc tag embedded within Fv-HSP72 on an automated immuno-staining system.
Distributional properties and homogeneity of variance were examined for all study variables to ensure that the assumptions for the selected analyses were supported. Student t tests were performed when comparing 2 groups. For multiple group comparisons, 1-way analysis of variance (ANOVA) was used with post hoc Bonferroni-corrected independent sample t tests. Values of p < 0.05 were considered significant, with Bonferroni correction applied for multiple comparisons. Differential treatment effects according to sample location (i.e., apex vs. AAR sans apex vs. remote region) were assessed with 2-way ANOVA testing the treatment × location interaction. All data are expressed as mean ± SD.
The Online Appendix provides additional information about the methods used.
Because Fv-HSP72 has already been shown to penetrate COS-7 cells and nondividing rat primary cortical neurons in vitro and protect them from H2O2-generated oxidative damage (15), we investigated its efficacy with human primary cardiomyocytes. Cells were seeded into opaque bottom 96-well tissue culture plates (5,000 cells/well) and allowed to adhere before replacement of the media with fresh CMM-prf media containing green dye–a cell impermeant dye whose fluorescence is dependent on DNA binding. Exposure to H2O2 induces oxidative stress and subsequent apoptosis (19), leading to increasing exposure of DNA to the cell-impermeant green dye and concomitant increases in fluorescent signal (Figure 2). Wells receiving 0.1 nmol of Fv-HSP72 30 min after the start of H2O2 intoxication had significantly lower cell death than untreated controls. This reduction in cell death was compromised if the 3E10-Fv were added in molar excess as a competitive inhibitor before Fv-HSP72. As expected, the same molar quantity of 3E10-Fv alone (100 nmol) used in the competitive inhibition experiment did not affect apoptosis. Adding a molar equivalent of HSP72 alone (0.1 nmol) also was not effective in reducing apoptosis compared with cells exposed to H2O2 only. Wells not intoxicated with H2O2 had very little cell death over the course of the experiments.
To determine the maximum signal achievable and calculate the percentage of cell death, some of these wells were exposed to lysis buffer and the values used to normalize the results. Whether cardiomyocyte intoxication included 12 h of H2O2 exposure (Figure 2A), overnight exposure (Figure 2B), or longer (in 1 study, up to 26 h; data not shown), Fv-HSP72 treatment inhibited cell death. Longer exposure of cells, either to a molar equivalent of HSP72 alone or a molar excess of 3E10-Fv alone, did not inhibit apoptosis. These observations reflected the in vivo results subsequently obtained.
Figure 3 presents representative in vivo and ex vivo μSPECT/μCT images of each of the 5 subgroups of animals. The simultaneous CT images allowed precise localization of the radiotracer uptake in the cardiac silhouette. More intense and more extensive 99mTc-annexin-V uptake was seen in the 3 control groups compared with the 2 Fv-HSP72 treatment groups (quantification discussed later). Maximum 99mTc-annexin-V uptake was observed predominantly in the apical region of the heart. The ex vivo μSPECT images of 99mTc-annexin-V myocardial uptake confirmed substantially lower radiotracer uptake in the Fv-HSP72–treated animals compared with control animals. SPECT/CT images in the axial, coronal, and sagittal planes depicted the extent of 99mTc-annexin-V uptake in the myocardium (Online Figure 1).
Such uptake was quantitatively evaluated by gamma counting of the harvested myocardial sections and expressed as %ID/g tissue. Figure 4 shows %ID/g uptake in the apex, AAR, and the remote area. P values displayed in the figure were computed from 3 pairwise comparisons of samples exposed to a control treatment (phosphate-buffered saline, 3E10-Fv alone, or HSP72 alone) versus samples exposed to the active treatment (Fv-HSP72 pre- or post-reperfusion) for each of the 3 locations using Bonferroni-corrected independent sample t tests (α = 0.01). Within each location, variance in %ID/g was similar between the active and control treatment groups. Average %ID/g uptake was significantly lower in the Fv-HSP72–treated groups versus control groups (phosphate-buffered saline, 3E10-Fv alone, and HSP72 alone) for samples collected from the apex (0.37 ± 0.18 vs. 0.68 ± 0.20; p = 0.0001), and AAR (0.28 ± 0.12 vs. 0.49 ± 0.15; p = 0.0003), and marginally lower in the noninfarcted region (0.05 ± 0.01 vs. 0.06 ± 0.02; p = 0.016). Analysis of the AAR included %ID/g data from the apex, given its location as a subregion of the AAR. We also analyzed the %ID/g for the AAR as 2 separate regions comprising the apex and those fragments in the AAR outside the apex. A 2-way ANOVA model suggested significant differences in %ID/g tissue uptake among the 5 study groups (p < 0.0001) and between the 3 different locations within the heart (i.e., apex vs. AAR sans apex vs. remote region; p < 0.0001). The size of the treatment effect differed across the sites (2-way ANOVA test of location × treatment interaction, p = 0.042).
Assessing infarct size
Figure 5 summarizes the cardiac troponin I concentration during the experiment. Independent sample t tests revealed significantly higher troponin I levels at 1 and 3 h post-reperfusion (data available for n = 25 animals, p = 0.043; and n = 21 animals, p = 0.041, respectively) for all 3 control animals versus the rabbits that received Fv-HSP72 pre- or post-reperfusion. Echocardiography was performed in the 3E10-Fv and HSP72 alone groups and the Fv-HSP72 post-reperfusion group. Figure 6 displays the sequential changes of LVEF in the control and active therapy groups. Repeated measures ANOVA reveals a significant time × treatment interaction for LVEF (p = 0.004). Paired comparisons with independent sample t tests at each time point revealed no difference in LVEF at baseline or during occlusion; however, during the post-reperfusion period, LVEF was significantly higher for samples with Fv-HSP72 treatment (n = 3) compared with the control (n = 12) groups (47.9 ± 3.7% vs. 37.6 ± 3.6%; p = 0.022). Visually, one could see recovery of the wall motion abnormality to a level closer to baseline during reperfusion in Fv-HSP72–treated rabbits (Online Figure 2) compared with 3E10-Fv and HSP72 alone control animals.
Myocardial tissue sections from the infarct zone, AAR, and remote myocardial region were examined from all 5 groups for histological, histochemical, and immunohistochemical characterization (Figure 7). Hematoxylin and eosin and Masson’s trichrome staining revealed myocyte necrosis with contraction bands in the infarct zone. There appeared to be greater interstitial hemorrhage in the infarcted tissues of control rabbits compared with those treated with Fv-HSP72. TUNEL staining to detect DNA fragmentation during apoptosis was more numerous in the hearts of control rabbits compared with Fv-HSP72–treated animals. A linear correlation was seen between TUNEL-positive nuclei and the %ID/g uptake of 99mTc-annexin-V in the heart fragments (p < 0.05; R2 = 0.5218). Finally, 3E10 localization with and without the HSP72 moiety was demonstrated by immunostaining for the c-myc tag with mouse monoclonal 9E10 seen as brown staining of the nucleus (arrows) in the cells. The HSP72 alone and phosphate-buffered saline control animals did not have a c-myc tag and, hence, no c-myc–positive brown nuclei (data not shown).
The present study found that a single intravenous injection of Fv-HSP72 had a significant myocardial protective effect in the rabbit model of ischemia-reperfusion injury. The extent of annexin uptake in μSPECT images and gamma-counting of myocardial tissue specimens within the AAR was significantly lower in the Fv-HSP72 treatment groups than in the 3 control groups, with a net reduction of 43% in myocardial damage. Also, the cardiac troponin I concentration was correspondingly lower with a net 42% reduction. LVEF was 27% higher in the Fv-HSP72 treatment groups compared with the control groups of animals.
Approaches under study
Although both necrosis and apoptosis play a significant role in cell death during ischemia and reperfusion, they constitute a continuum of myocardial injury. The ischemic injury is initiated by the energy-requiring process of apoptosis. However, because the ischemic cells are devoid of energy, the cells die of necrosis. Therefore, it is expected that an intervention targeted at any component of myocardial injury would reduce net cell death (20). Institution of reperfusion contributes to resolution of apoptosis within the AAR; ironically, it also imposes unintended adverse effects on myocardial cells (reperfusion injury) (21).
The ultimate treatment goal for patients with acute coronary syndrome is to achieve early reperfusion and resolution of ischemia by definitive intervention (e.g., percutaneous coronary intervention) or at least postpone cell death to allow subsequent definitive intervention if it is not available instantly (22). For the latter, thrombolytic therapy with pharmacological aids becomes important and is particularly important in low- and middle-income countries (23).
Multiple adjunctive pharmacological approaches have been proposed to reduce infarct size in animal models and clinical studies of ischemia-reperfusion injury, but they have shown minimal success (3,4). A large randomized study found only a 2.2% increase in ejection fraction, using continuous infusion of atrial natriuretic peptide in patients with acute myocardial infarction, and 29 (10.5%) of 277 patients in the atrial natriuretic peptide group had severe hypotension (24). Similarly, in another trial of 410 patients, a single intravenous cyclosporine bolus just before primary percutaneous coronary intervention had no effect on ST-segment resolution or high-sensitivity cardiac troponin T and did not improve clinical outcomes or LV remodeling up to 6 months (25). Conversely, there is renewed interest in beta-1 selective blockers, and multicenter studies are underway (26).
In patients with anterior ST-segment elevation myocardial infarction with Killip class I or II undergoing primary percutaneous coronary intervention, early intravenous metoprolol (delivered in the ambulance) before reperfusion resulted in higher long-term LVEF, a reduced incidence of severe LV systolic dysfunction and implantable cardioverter-defibrillator indications, and fewer heart failure admissions. In addition to the pharmacological approaches, various biologic agents are being evaluated in experimental studies, such as those targeting apoptotic cascades through caspase (22) and Ca(2+)-calmodulin–dependent protein kinase (27) pathways. Alternatively, fortification of protective biologic agents, such as Fv-HSP72, has shown beneficial results in cerebral infarction (13).
More than 3 decades of research have demonstrated the cardioprotective effects of HSP72 in both in vitro and in vivo investigations. Expression of HSP72 is increased by elevated temperature or ischemic stimuli, gene transfection (28), and transgenic overexpression (29). HSP72 is a pleiotropic cytoprotectant, not only in the cardiovascular system but also in other organs, facilitating intracellular folding or unfolding, transportation, and chaperoning of proteins (30). HSP72 has also been implicated in the disruption of 3 apoptotic pathways: 1) adenosine triphosphate–dependent (the apoptosome pathway) (31); 2) adenosine triphosphate–independent (apoptosis-inducing factor pathway) (32); and 3) the nuclear factor-κB pathway (33).
HSPs are largely intracellular proteins, and their administration is not associated with any reported cytotoxicity. Because the cardioprotective effects of HSP72 have been demonstrated, even small molecules to induce transcription have also been used (34). However, this approach has 2 limitations. First, both stimuli and gene therapy for HSP induction have a time lag of >3 h before reaching therapeutic levels; this time frame precludes their use as treatment for acute coronary syndromes. Second, the endogenous induction of HSP is reportedly less responsive with increasing age (35,36).
3E10-Fv-HSP72 fusion protein
The present study circumvents the HSP72 induction time lag by using an ENT2 transporter–mediated 3E10-Fv–directed system to deliver therapeutic protein into living cells (Central Illustration). 3E10-Fv, a candidate antibody for protein delivery, is the single-chain fragment of a murine anti-DNA autoantibody 3E10 (37). It consists of the variable region of both the light and heavy chains of monoclonal antibody 3E10 fused with a short (GGGGS)3 linking sequence. 3E10-Fv penetrates cells through the energy-independent ENT2 channel, which is located in both the plasma and nuclear (38) membranes. The target selectivity of 3E10 in vivo is based on the simple concept that tissues undergoing significant cell injury possess a high concentration of extracellular DNA. Binding to DNA is necessary for 3E10 passage (12). Salvaging of nucleosides by surrounding cells, through the ENT2 channel, provides 3E10 the opportunity to enter the injured cells. The toxicity of Fv-HSP72 is negligible because it is produced by stable expression of Fv complementary DNA in Chinese hamster ovary cell lines (37); after localizing in the cell nucleus, 3E10-Fv is largely degraded within 4 h.
The antibody 3E10-Fv has been widely used to deliver numerous potentially therapeutic peptides or proteins into living cells, such as p53 for cancer and micro-dystrophin for muscular dystrophy (39). In a previous study using a rat ischemic brain model, injection of Fv-HSP72 noticeably reduced infarct volume (68%) and improved sensorimotor function (13). In the present study, we demonstrated the cardioprotective effects of Fv-HSP72 in a myocardial ischemia-reperfusion model. Immunostaining for the c-myc tag incorporated into Fv-HSP72 demonstrated penetration of the molecule into cardiomyocytes within the infarct zone, an important observation to counter the suggestion that the effects were due to induction of HSPs caused by the stress of ischemia and reperfusion. The administration of HSP72 or 3E10-Fv alone did not reduce annexin-V myocardial uptake and had no effect on functional recovery. These data were consistent with the hypothesis that Fv-HSP72, not HSP72 alone, can penetrate the cell membrane and acts as a cardioprotective agent.
Based on messenger ribonucleic acid levels surveyed in the organs of mice and rats, ENT2 tissue distribution varies, with the heart and brain showing greater amounts than some other organs. ENT2 is also an energy-independent channel through which molecules can readily pass down the concentration gradient between the extracellular and intracellular compartments. This scenario means that energy-deficient but potentially viable ischemic cells, as seen in reperfusion injury, can still take up 3E10-Fv even when endocytosis and transporters cease to function. It can be further hypothesized that a minimal increase in the normal cells might render them more resistant to damage.
Molecular imaging of apoptotic cells
Annexin-V, a human protein with a molecular weight of 36 kDa, targets the expression of phosphatidylserine on the outer leaflet of the cell membrane in apoptotic cells (14,40). Phosphatidylserine is a constitutive plasma membrane anionic phospholipid that is actively recruited to the inner leaflet of the lipid bilayer. Phosphatidylserine is virtually absent from the outer surface of normal cells (41). After injecting 99mTc annexin-V, one can identify the site and extent of severely stressed and apoptotic cells, and gamma counting allows quantitative assessment of uptake within each cross-section of the heart. Our data confirmed the cardioprotective effects of Fv-HSP72, correlating a substantially lower radiotracer uptake in μSPECT imaging and gamma counting of myocardial tissue segments compared with control groups. Histological analysis of the myocardium further illustrated the tissue protection afforded by the Fv-HSP72 treatment. TUNEL staining detects DNA fragmentation during apoptosis, and those sections of infarcted tissue from Fv-HSP72 rabbits had fewer TUNEL-positive nuclei than control rabbits. Furthermore, there appeared to be less interstitial hemorrhage in Fv-HSP72–treated rabbits. In addition, the echocardiographic data indicated functional salvage. These data show that the acute delivery of Fv-HSP72, but not HSP72 alone, could at least partially resolve myocardial ischemia-reperfusion injury.
The cardiac troponin I measurement and LV functional assessment indicated that Fv-HSP72 reduced the infarct volume and improved LV functional recovery after an ischemia-reperfusion injury. Echocardiographic LV function and cardiac troponin levels are not perfect measurements of infarct size; however, the modified trichrome staining with scintigraphy evidence offer significant insight into infarct size. Although triphenyl tetrazolium chloride staining of heart specimens is often used as a means of visualizing infarct size, conventional histopathological assessments are an acceptable alternative, especially for shorter reperfusion times (42). These assessments each provided data supporting the hypothesis that administration of Fv-HSP72 before or after reperfusion reduced myocyte cell death and infarct size.
First, the effect of Fv-HSP72 was assessed only in the acute phase. Subacute and chronic effects of Fv-HSP72 need to be investigated with longer intervals of follow-up to determine the durability of this acute salvage. Second, dose dependency and frequency of Fv-HSP72 injection were not addressed in the present study. Future studies with a larger animal model would clarify them.
A single injection of Fv-HSP72 reduced myocardial apoptosis and improved LV function in an ischemia-reperfusion model of acute myocardial injury in rabbits. In contrast, injection of HSP72 or 3E10-Fv alone showed no evidence of cardioprotection, although 3E10-Fv is capable of transporting HSP72 into stressed, but potentially salvageable, cardiomyocytes. Fv-HSP72 needs further investigation in larger animals and in prospectively designed randomized clinical trials.
COMPETENCY IN MEDICAL KNOWLEDGE: Rapid delivery of HSP72 by using 3E10-Fv provided cardioprotective effects in a rabbit ischemia-reperfusion model. The biologics-based drugs are likely to play a greater role in disease management.
TRANSLATIONAL OUTLOOK: Rapid delivery of HSP72 using the 3E10-Fv carrier may be useful as an adjunct to reperfusion therapy for the treatment of acute myocardial infarction. Fv-HSP72 needs further investigation in larger animals and in prospectively designed randomized clinical trials.
For supplemental Methods and figures, please see the online version of this article.
This work was supported by a National Institutes of Health/National Heart, Lung, and Blood Institute grant (R43HL122012-01A1). Dr. Tanimoto was supported by Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad. Dr. Nakahara was supported by Uehara Memorial Foundation and SNMMI Wagner-Torizuka Fellowship. Dr. Parseghian, Mr. Richieri, and Dr. Reynolds declare they are owners of Rubicon Biotechnology, which is developing the Fv-HSP72 technology. Dr. Billimek does statistical consulting for Rubicon; however, he is not an employee nor does he have any ownership in the company. Dr. Nishimura, Dr. Weisbart, and Ms. Chan are employees of UCLA and the VA. They also have no ownership in Rubicon. Dr. Sengupta is an advisor to Heart Test Laboratories and Hitachi Aloka. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Tanimoto and Parseghian contributed equally to this work. P.K. Shah, MD, served as Guest Editor-in-Chief for this paper.
- Abbreviations and Acronyms
- micro–computed tomography
- micro–single-photon emission computed tomography
- percent injected dose per gram
- area at risk
- analysis of variance
- deoxyribonucleic acid
- equilibrative nucleoside transporter 2
- single-chain fragment variable
- hydrogen peroxide
- heat shock protein
- left ventricular
- left ventricular ejection fraction
- terminal deoxynucleotidyl transferase dUTP nick end labeling
- Received April 3, 2017.
- Revision received June 6, 2017.
- Accepted July 17, 2017.
- 2017 American College of Cardiology Foundation
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