**3.2 Animal experiment in acute ischemia and reperfusion**

In a mouse model of 15 and 30 min of ischemia and 90 min of reperfusion, percentage of cardiomyocytes staining positivity for biotinylated annexin V was far greater than the

Apoptosis Imaging in Diseased Myocardium 257

1.47±0.11, respectively) and only mild uptake was observed at 6 h and 24 h after reperfusion. Interestingly, no morphological sign of necrosis and apoptosis was observed until 24 h after reperfusion in 10 min ischemia and only micro foci of cell degeneration and cell infiltration were observed until 24 h after reperfusion in 15 min ischemia, with mild TUNEL positivity which peaked at 6 hr (1.03±0.40% in 10 min ischemia and 2.84±0.94% in 15 min ischemia). These data indicate that 99mTc-annexin V uptake can be observed after mild ischemic insults

Several cardioprotective interventions such as ischemic preconditioning, postconditioning, and caspase inhibitor preparation attenuate 99mTc-annexin V uptake (Taki, et al., 2007). Ischemic preconditioning suppressed 99mTc-annexin V binding by 90 % of control and

Reversibility of PS externalization in brief ischemia was demonstrated (Kenis, et al., 2010). In murine myocardium with 5 min ischemia and 90 min and 6 h of reperfusion, fluorescent labeled annexin V injected before 10 min of sacrifice binds cell surface, but not at 24 h after reperfusion. On the other hand fluorescent-annexin V injected at the onset of reperfusion exclusively localized intracellularly at 90 min, 6 h, and 24h after reperfusion. No TUNEL positivity was observed in this 5 min ischemia and reperfusion model. These data indicate that in brief ischemia, PS externalizes transiently, which is amenable to targeting by annexin V for at least 6 h after reperfusion, and internalize after annexin V binding (reversibility of

In human pathological study by TUNEL staining and DNA electrophoresis, widespread apoptosis in infarcts was observed only a few hours in age before the appearance of coagulative necrosis (Veinot, et al. 1997). In addition, TUNEL positibity was observed primarily in myocytes containing contraction bands, which occur predominantly in regions of reperfused myocardium. These findings are consistent with animal experiment, and in infarcted human myocardium, apoptosis is the early and predominant form of cell death

First clinical imaging with 99mTc-annexin V in 7 patients with acute myocardial infarction demonstrated significant uptake of 99mTc-annexin V (injected 2 h after reperfusion) in the area corresponding to the perfusion defect in 6 patients (Hofstra, et al., 2000). Subsequent study in 9 patients with acute myocardial infarction, 99mTc-annexin V uptake (injected within 1.5 - 7 h after reperfusion) was again clearly visualized on SPECT performed at 15 h later in infracted areas with a matching perfusion defect confirmed by 99mTc-sestamibi SPECT before reperfusion. Repeat 99mTc-sestamibi SPECT at 1 – 3 week after the onset of infarction demonstrated that the perfusion defects were significantly smaller than the defects in acute phase, suggesting that the significant amount of myocardium in perfusion defect with 99mTcannexin V uptake in acute phase might be in reversible damage rather than irreversible necrosis. Imaging of the extent of apoptosis resulting from acute coronary syndromes could be a valuable tool to help guide revascularization strategies and therapy with anti-apoptotic drugs if available in near future. Other potential application of 99mTc-annexin V imaging would be in the detection of myocardial ischemic insults in patients with acute coronary syndrome without ST elevation and troponin leakage, in assessment of the effect of revascularization in acute coronary syndrome, in evaluation of the effect of postconditioning at reperfusion therapy, in detection of transient PS exetrnalization due to brief spontaneous

postoconditioning and caspase inhibitor attenuate the binding by around 70 %.

those do not result in myocardial infarction.

**3.3 Clinical findings and potential future application** 

and its appearance is accelerated in reperfused myocardium.

the apoptotic process).

percentage staining positivity for IgG, which indicates plasma cell membrane leakage (Dumont et al., 2000). Accordingly, at least 90 min of reperfusion, most of the annexin V uptake might represent PS externalization.

We investigated the temporal and spatial change of 99mTc-annexin V accumulation in a rat model of 20 min coronary artery occlusion and reperfusion. 99mTc-annexin V uptake was imaged at 30 min, 90 min, 6 h, 24 h, 3 day, 2 weeks after reperfusion. The strongest 99mTcannexin V uptake was observed in the mid myocardium of the area at risk at 30 m and 90 m of reperfusion and the uptake expanded to subendocardial and subepicardial layer at 6 h after reperfusion, followed by gradual reduction of the uptake over 3 days (Fig. 2) (Taki, et al., 2004). On the other hand, TUNEL positivity peaked at 6 h to 1 day after reperfusion. These findings are in keeping with the known temporal sequence of apoptosis, in which one of the earliest events is externalization of phosphatidylserine, followed by DNA fragmentation. Using fluorescent labeled annexin V, real time monitoring of annexin V binding to cardiomyocytes after 30 m ischemia and reperfusion demonstrated that cardiomyocytes started to bind annexin V within minute and the amount of binding reached a maximum within 20-25 m (Dumont, et al., 2001). These finding indicate that apoptosis commences just after ischemia and reperfusion and PS externalization reaches plateau around 30 m after reperfusion.

Fig. 2. Autoradiography of 99mTc-annexin V and 201Tl at various reperfusion time after 20 min of coronary artery occlusion.

At a examination time after 20 min ischemia and reperfusion, 99mTc-annexin V (80-150 MBq) was injected and 1 h later 201Tl (0.74 MBq) was injected just after the coronary artery reocclusion to verify the area at risk. Upper low demonstrated 99mTc-annexin V images and lower low shows 201Tl image that represents area at risk.

The amount of annexin V binding to apoptotic cardiomyocytes after reperfusion depends on the severity of ischemia and reperfusion time (Taki, et al., 2007a). In a rat model of 5 min, 10 min, 15 min ischemia and reperfusion model, degree of 99mTc-annexin V accumulation depend on the length of the coronary artery occlusion time and time period after reperfusion: significant uptake of 99mTc-annexin V accumulation was observed in 15 min and 10 min ischemia (uptake ratio was 4.46±3.16 and 2.02±0.47, respectively), whereas, no significant uptake was observed in 5 min ischemia at 30 min after reperfusion. 99mTc-annexin V uptake in 15 min and 10 min ischemia reduced at 90 min (uptake ratio was 3.49±1.78 and

percentage staining positivity for IgG, which indicates plasma cell membrane leakage (Dumont et al., 2000). Accordingly, at least 90 min of reperfusion, most of the annexin V

We investigated the temporal and spatial change of 99mTc-annexin V accumulation in a rat model of 20 min coronary artery occlusion and reperfusion. 99mTc-annexin V uptake was imaged at 30 min, 90 min, 6 h, 24 h, 3 day, 2 weeks after reperfusion. The strongest 99mTcannexin V uptake was observed in the mid myocardium of the area at risk at 30 m and 90 m of reperfusion and the uptake expanded to subendocardial and subepicardial layer at 6 h after reperfusion, followed by gradual reduction of the uptake over 3 days (Fig. 2) (Taki, et al., 2004). On the other hand, TUNEL positivity peaked at 6 h to 1 day after reperfusion. These findings are in keeping with the known temporal sequence of apoptosis, in which one of the earliest events is externalization of phosphatidylserine, followed by DNA fragmentation. Using fluorescent labeled annexin V, real time monitoring of annexin V binding to cardiomyocytes after 30 m ischemia and reperfusion demonstrated that cardiomyocytes started to bind annexin V within minute and the amount of binding reached a maximum within 20-25 m (Dumont, et al., 2001). These finding indicate that apoptosis commences just after ischemia and reperfusion and PS externalization reaches plateau

Fig. 2. Autoradiography of 99mTc-annexin V and 201Tl at various reperfusion time after 20

was injected and 1 h later 201Tl (0.74 MBq) was injected just after the coronary artery reocclusion to verify the area at risk. Upper low demonstrated 99mTc-annexin V images and

At a examination time after 20 min ischemia and reperfusion, 99mTc-annexin V (80-150 MBq)

The amount of annexin V binding to apoptotic cardiomyocytes after reperfusion depends on the severity of ischemia and reperfusion time (Taki, et al., 2007a). In a rat model of 5 min, 10 min, 15 min ischemia and reperfusion model, degree of 99mTc-annexin V accumulation depend on the length of the coronary artery occlusion time and time period after reperfusion: significant uptake of 99mTc-annexin V accumulation was observed in 15 min and 10 min ischemia (uptake ratio was 4.46±3.16 and 2.02±0.47, respectively), whereas, no significant uptake was observed in 5 min ischemia at 30 min after reperfusion. 99mTc-annexin V uptake in 15 min and 10 min ischemia reduced at 90 min (uptake ratio was 3.49±1.78 and

uptake might represent PS externalization.

around 30 m after reperfusion.

min of coronary artery occlusion.

lower low shows 201Tl image that represents area at risk.

1.47±0.11, respectively) and only mild uptake was observed at 6 h and 24 h after reperfusion. Interestingly, no morphological sign of necrosis and apoptosis was observed until 24 h after reperfusion in 10 min ischemia and only micro foci of cell degeneration and cell infiltration were observed until 24 h after reperfusion in 15 min ischemia, with mild TUNEL positivity which peaked at 6 hr (1.03±0.40% in 10 min ischemia and 2.84±0.94% in 15 min ischemia). These data indicate that 99mTc-annexin V uptake can be observed after mild ischemic insults those do not result in myocardial infarction.

Several cardioprotective interventions such as ischemic preconditioning, postconditioning, and caspase inhibitor preparation attenuate 99mTc-annexin V uptake (Taki, et al., 2007). Ischemic preconditioning suppressed 99mTc-annexin V binding by 90 % of control and postoconditioning and caspase inhibitor attenuate the binding by around 70 %.

Reversibility of PS externalization in brief ischemia was demonstrated (Kenis, et al., 2010). In murine myocardium with 5 min ischemia and 90 min and 6 h of reperfusion, fluorescent labeled annexin V injected before 10 min of sacrifice binds cell surface, but not at 24 h after reperfusion. On the other hand fluorescent-annexin V injected at the onset of reperfusion exclusively localized intracellularly at 90 min, 6 h, and 24h after reperfusion. No TUNEL positivity was observed in this 5 min ischemia and reperfusion model. These data indicate that in brief ischemia, PS externalizes transiently, which is amenable to targeting by annexin V for at least 6 h after reperfusion, and internalize after annexin V binding (reversibility of the apoptotic process).

#### **3.3 Clinical findings and potential future application**

In human pathological study by TUNEL staining and DNA electrophoresis, widespread apoptosis in infarcts was observed only a few hours in age before the appearance of coagulative necrosis (Veinot, et al. 1997). In addition, TUNEL positibity was observed primarily in myocytes containing contraction bands, which occur predominantly in regions of reperfused myocardium. These findings are consistent with animal experiment, and in infarcted human myocardium, apoptosis is the early and predominant form of cell death and its appearance is accelerated in reperfused myocardium.

First clinical imaging with 99mTc-annexin V in 7 patients with acute myocardial infarction demonstrated significant uptake of 99mTc-annexin V (injected 2 h after reperfusion) in the area corresponding to the perfusion defect in 6 patients (Hofstra, et al., 2000). Subsequent study in 9 patients with acute myocardial infarction, 99mTc-annexin V uptake (injected within 1.5 - 7 h after reperfusion) was again clearly visualized on SPECT performed at 15 h later in infracted areas with a matching perfusion defect confirmed by 99mTc-sestamibi SPECT before reperfusion. Repeat 99mTc-sestamibi SPECT at 1 – 3 week after the onset of infarction demonstrated that the perfusion defects were significantly smaller than the defects in acute phase, suggesting that the significant amount of myocardium in perfusion defect with 99mTcannexin V uptake in acute phase might be in reversible damage rather than irreversible necrosis. Imaging of the extent of apoptosis resulting from acute coronary syndromes could be a valuable tool to help guide revascularization strategies and therapy with anti-apoptotic drugs if available in near future. Other potential application of 99mTc-annexin V imaging would be in the detection of myocardial ischemic insults in patients with acute coronary syndrome without ST elevation and troponin leakage, in assessment of the effect of revascularization in acute coronary syndrome, in evaluation of the effect of postconditioning at reperfusion therapy, in detection of transient PS exetrnalization due to brief spontaneous

Apoptosis Imaging in Diseased Myocardium 259

1997; Saraste, et al., 1999; Guerra, et al., 1999). In study with transgenic mice that express a conditionally active caspase exclusively in the myocardium, low rate of cardiomyocyte apoptosis as 0.023% is sufficient to cause a lethal dilated cardiomyopathy. Conversely, inhibition of cardiac myocyte apoptosis by caspase inhibitor in this murine model largely prevents the development of cardiac dilation and contractile dysfunction, indicating that myocyte apoptosis may be a causal mechanism of heart failure, and inhibition of this cell death process may constitute the basis for novel therapies (Wencker, et al., 2003). The low level of cell death due to apoptosis in heart failure makes detection of apoptosis with the

In recent study in 9 consecutive patients with advanced nonischemic cardiomyopathy (8 dilated and 1 hypertrophic cardiomyopathy) and 2 relatives, 5 patients showed focal or global 99mTc-annexin V uptake in the left ventricular myocardium. Interestingly, these 5 patients with 99mTc-annexin V uptake had experienced a significant worsening or a recent onset of heart failure, on the contrary, 4 patients without 99mTc-annexin V uptake had no recent evidence of worsening of heart failure. In addition, during a follow up of 1 year, 4 patients with 99mTc-annexin V uptake showed a decline of LVEF, on the other hand, in patients without 99mTc-annexin V uptake, clinical status and LVEF remained stable (Kietselaer, et al., 2007). These data indicate that the 99mTc-annexin V imaging in advanced non-ischemic cardiomyopathy, may identify the patients with high risk who might benefite

In a rat model of autoimmune myocarditis, 99mTc-annexin V (HYNIC annexin V) and 14Cdeoxyglucose (DG) uptakes were examined (Tokita, et al.,2003). Myocarditis was triggered by an immunization of rats by infusing porcine cardiac myosine and the rats formed antibodies against the myosin and developed myocarditis. In acute phase of myocarditis, both 99mTc-annexin V (2.8 time more than normal rats) and 14C-DG (2.7 time more than normal rats) uptake increased significantly, however, only 99mTc-annexin V distribution correlated with the TUNEL positive area, and the distribution of 14C-DG correlated with inflammatory cell infiltration. In subacute phase, 99mTc-annexin V uptake returned normal level, on the other hand, 14C-DG uptake decreased but still higher uptake reflecting prolonged mild inflammatory cell infiltration. In this model of immune myocarditis, there was a marked difference in distribution of apoptotic cell death and inflammation. The data indicates that 99mTc-annexin V uptake is specific in myocardial apoptotic process induced by

Another animal experiment in a rat model that develops spontaneous myocarditis mimicking catecholamine induced subacute myocarditis demonstrated significantly increased 99mTc-annexin V uptake in planar scinitgraphy. Autoradiogrpahy confirmed increased 99mTc-annexin V uptake. Histopatology demonstrated patchy areas of interstitial edema with inflammatory cells in the perivascular areas and at cardiocyte layers, and myocyte necrosis with nuclear extrusion, scattered throughout the myocardium and apostatin-positive cells were diffusely but inhomogeneously distributed throughout the

In myocarditis, apoptosis imaging might play an important role, in confirming the diagnosis in terms of the extent of the involvement and disease activity, selecting patients with

current techniques very challenging.

from cell death blocking therapies.

myocardium (Peker, et al., 2004).

**5. Apoptosis imaging in myocarditis** 

inflammation and is independent of inflammatory cell infiltration.

antiapoptotic therapy and monitoring the effect of therapy.

or stress induced ischemia. However, several questions yet to be answered concerning the clinical application of 99mTc-annexin V imaging. In vivo imaging 1 hr after tracer injection is feasible in rats because blood clearance of 99mTc-annexin V was rather fast, although, the earliest optimal time for imaging after tracer injection should be investigate in human. In heart transplantation patients, it was revealed that 99mTc-annexin V SPECT imgaing was possible 1 h after tracer injection (Narula, et al., 2001). Speedy imaging after tracer injection is crucial especially in emergency situation. Are therapeutic interventions beneficial for all annexin-positive myocardium, or in only specific pathological status, or in only limited time window? How much of the shift from necrosis to apoptosis by therapeutic intervention is cardioprotective (Narula, et al., 2003)? Necrosis is more harmful than apoptosis, because cells are removed without inflammation in apoptosis but cells are removed with inflammation and fibrosis follows in necrosis. Is annexin V positive scan in stress induced ischemia related to subsequent prognosis, or indication of PCI? These potential imaging concepts of the assessment of myocardial injury, stress, cell death in acute ischemia should be validated in clinical studies.

It has been increasingly clear that apoptosis is a major contributor to early cardiomyocyte cell death after acute myocardial infarction, and is involved in post myocardial infarction ventricular dysfunction and adverse remodeling that develop heart failure. These findings emphasize the need for the reliable in-vivo imaging of apoptosis that assess the ongoing pathology so that rational preventive therapies can be applied and to assess the consequence of therapies. 99mTc-annexin V imaging might be applied to assess myocardium at risk or cell death in acute coronary syndrome and prediction of the ventricular remodeling after myocardial infarction and heart failure by allowing visualization of ongoing PS externalization that might precede or underlie change in pathophysiology, morphology, and LV dysfunction.
