The TIME Trial - Effect of Timing of Stem Cell Delivery Following ST-Elevation Myocardial Infarction on the Recovery of Global and Regional Left Ventricular Function: Final 2-Year Analysis

Abstract

Rationale

The TIME trial was the first cell therapy trial sufficiently powered to determine if timing of cell delivery following ST-elevation myocardial infarction (STEMI) affects recovery of left ventricular (LV) function.

Objective

To report the 2-year clinical and cardiac magnetic resonance imaging (cMRI) results and their modification by microvascular obstruction (MVO).

Methods and Results

TIME was a randomized, double-blind, placebo-controlled trial comparing 150 million bone marrow mononuclear cells (BMC) vs. placebo in 120 patients with anterior STEMIs resulting in LV dysfunction. Primary endpoints included changes in global (LV ejection fraction (LVEF)) and regional (infarct and border zone) function. Secondary endpoints included changes in LV volumes, infarct size and major adverse cardiac events. Here, we analyzed the continued trajectory of these measures out to 2 years and the influence of MVO present at baseline on these long-term outcomes. At 2 years (n=85), LVEF was similar in the BMC (48.7%) and placebo groups (51.6%) with no difference in regional LV function. Infarct size and LV mass decreased ≥ 30% in each group at 6-months and declined gradually to 2-years. LV volumes increased approximately 10% at 6-months and remained stable to 2-years. MVO was present in 48 patients at baseline and was associated with significantly larger infarct size (56.5 g vs. 36.2 g), greater adverse LV remodeling, and marked reduction in LVEF recovery (0.2 vs. 6.2).

Conclusions

In one of the longest serial cMRI imaging analyses of patients with large anterior STEMIs, BMC administration did not improve recovery of LV function over 2-years. MVO was associated with reduced recovery of LV function, greater adverse LV remodeling and more device implantations. The use of cardiac MRI leads to greater drop-out of patients over time due to device implantation in patients with more severe LV dysfunction resulting in overestimation of clinical stability of the cohort.

Clinical Trial Registration

NCT00684021 [https://clinicaltrials.gov/ct2/show/NCT00684021].

INTRODUCTION

The Cardiovascular Cell Therapy Research Network (CCTRN) was established by the National Heart, Lung and Blood Institute (NHLBI) to foster the development of adult stem cell-based trials in the United States.¹ An initial focus of the Network was the use of bone marrow mononuclear cells (BMCs) in the setting of ST-elevation myocardial infarction (STEMI) based on results suggesting benefit in left ventricular ejection fraction (LVEF) from several European trials.^{2, 3} An important question not addressed in these early trials was whether timing of cell delivery following reperfusion by primary percutaneous coronary intervention (PCI) with stenting influenced recovery of LV function, since temporal changes in the myocardium known to occur shortly following reperfusion could both promote or inhibit cell survival and engraftment.^{4, 5}

Accordingly, the CCTRN developed two trials, Timing in Myocardial Infarction Evaluation (TIME) and LateTIME, to investigate the role of timing of cell delivery following STEMI. Specifically, the TIME trial⁶ examined cell delivery on Day 3 versus Day 7 following primary PCI with stenting, while LateTIME⁷ examined if cell delivery 2–3 weeks post-STEMI could enhance the recovery of LV function. Although TIME found that BMCs did not improve the recovery of global or regional LV function compared to placebo at its 6-month primary endpoint, we pre-specified that patients were to be followed for 2-years with additional serial cardiac magnetic resonance imaging (cMRI) at 1- and 2-years. Here we report the final 2-year results for TIME that provides one of the largest serial cMRI measurements of patients with moderate to large anterior infarctions and demonstrates the powerful predictive effects of microvascular obstruction (MVO) on LV function when observed on the baseline MRI scans.

METHODS

Result data at the 6-month primary endpoint is available at clinicaltrials.gov and additional data from later time points can be requested from CCTRN Data Coordinating Center (lemmoye@msn.com).

Eligibility criteria of the TIME trial have been previously described in detail.⁸ In brief, TIME was a randomized, placebo-controlled trial of patients with anterior STEMI who underwent successful primary PCI with stenting and who had at least moderate left ventricular dysfunction (LVEF ≤45%) by screening echocardiography 1–2 days post-PCI. All patients provided informed written consent. The study was approved by the Institutional Review Boards of each participating center. Patients were randomized (1:1) to study product delivery on Day-3 or Day-7 post-PCI. On the day of delivery, patients underwent measurement of global and regional LV function and infarct size with a 1.5 T cardiac MRI scanner using protocols developed by the MRI Core Laboratory (University of Florida). Those patients randomized to Day-7 also had a cMRI performed on Day-3 to establish the same baseline between all patients. For the purposes of this paper, Day-3 images are heretofore referenced as baseline. All patients were prescribed standard post-MI medications including beta-blockers and ACE inhibitors (Table 1) that were recommended to be continued over the 2 years of follow-up. However, follow-up medications were not recorded. Data are:

Table 1.

Baseline and Cell Characteristics for TIME 2-Year Cohort (N=85)

	BMC N=58	Placebo N=27	P-Value
Patient Characteristics
Age mean (SD)	55.9 (11.0)	56.4 (10.4)	0.845
Female N (%)	7 (12)	4 (14)	0.737
Race
White N (%)	51 (87)	23 (85)	0.737
Nonwhite N (%)	7 (12)	4 (14)
Hispanic N (%)	2 (3)	1 (3)	1.000
History N (%)
Diabetes	7 (12)	5 (18)	0.508
Hypertension	26 (44)	22 (81)	0.002
Hyperlipidemia	41 (70)	20 (74)	0.802
Angina	8 (13)	5 (18)	0.747
Smoking	33 (56)	16 (59)	1.000
Physical mean (SD)
BMI	30.6 (5.6)	31.1 (5.4)	0.732
Ejection Fraction (EF)
Echo Screening	37.1 (6.3)	37.7 (4.8)	0.654
cMRI Core (3 day) EF	45.9 (9.4)	46.9 (8.7)	0.657
Medications N (%)
ACE inhibitor	52 (89)	21 (77)	0.184
Plavix/Prasugrel	55 (94)	27 (100)	0.548
Aspirin	55 (94)	27 (100)	0.548
Beta Blockers	56 (96)	27 (100)	1.000
Statins	52 (89)	26 (96)	0.423
Diuretics	12 (20)	6 (22)	1.000
Coumadin/Wararin/Lovenox	14 (24)	2 (7.)	0.080
Labs [N] mean (SD)
Hemoglobin	[50] 14.2 (1.5)	[21] 13.2 (2.1)	0.031
hsCRP	[53] 38.7 (48.8)	[25] 38.4 (29.6)	0.976
Peak CK	[48] 3210.2 (2178.6)	[25] 2027.1 (1820.4)	0.023
Peak CKMB [	[44] 258.3 (175.9)	[23] 185.8 (170.8)	0.111
BNP reg	[48] 293.3 (613.7)	[24] 286.9 (330.5)	0.962
BNP pro	[8] 1537.2 (1518.7)	[3] 2123.3 (3030.9)	0.669
MI Treatment
Ischemic Time hrs mean (SD)	6.4(9.6)	7.2 (7.4)	0.726
Time from Aspirate to Infusion (hrs) mean (SD)	8.9 (3.3)	8.4 (1.1)	0.450
Drug Eluting Stent N (%)	46 (79)	23 (85)	0.766
Cell Processing [N] mean × 106 (SD)
Final Cell Dose	[58] 146.1 (20.1)	[27] 148.9 (3.2)	0.471
Viability [N] mean (SD)	[58] 98.1 (1.6)	[27] 98.2 (1.5)	0.862
CD34+ ISHAGE	[53] 2.0 (1.1)	[26] 2.4 (1.0)	0.184
CD133+ ISHAGE	[53] 1.0 (0.6)	[26] 1.3 (0.8)	0.088
CFUec per dose	[41] 303.7 (479.6)	[18] 381.5 (812.3)	0.647
ECFC per dose	[41] 615.4 (1806.7)	[16] 163.5 (262.5)	0.326
MSC per dose	[40 ]511.9 (556.8)	[18] 364.6 (459.9)	0.331

BMC=bone marrow cells; SD=standard deviation; BMI=body mass index; ACE=angiotensin-converting enzyme; hsCRP=high sensitivity c-reactive protein; CK=creatine kinase; CKMB=creatine kinase myocardial band; BNP=brain natriuretic peptide; CFU=colony forming units; ECFC=endothelial colony forming cells; MSC=mesenchymal stem cells

Assessment of Microvascular Obstruction (MVO)

The presence of MVO (late) was identified using delayed gadolinium imaging in the baseline images. Images were acquired 15–20 minutes after the administration of gadolinium-DTPA (0.20 mmol/kg). Quantification of infarct tissue and MVO was performed using cvi42 post-processing software (Version 5.1; Calgary, AB, Canada). MVO was defined as an area of hypointensity within subendocardial hyperintensities. MRI analyses were performed at the Core Lab and readers were blinded to patient data and treatment assignment.

Cell processing and delivery

Following baseline cMRI, patients underwent a bone marrow aspiration and BMCs were isolated at each center using an automated, Ficoll-based cell separation device previously validated by the CCTRN for BMCs (Sepax, Biosafe Inc.).⁹

All patients randomized to BMCs received 150 million nucleated cells containing 70–80% BMCs. Patients randomized to placebo received a cell-free product of 5% albumin in normal saline with the addition of 100 uL of whole blood to maintain similar appearance to the BMC product.

Patients underwent intracoronary infusion of BMCs or placebo in the cardiac catheterization laboratory, within 12 hours of bone marrow aspiration, using the stop-flow technique.

Primary endpoints

The primary endpoints were changes in global LVEF and regional (infarct and border zone) LV function measured at 6-months by cMRI compared to baseline and whether day of treatment (Day-3 vs. Day-7) affected these results. Secondary endpoints were major adverse cardiac events and changes in LV volumes and infarct size.

Statistical methods

Baseline comparisons were conducted using two-sample unpaired t-tests for continuous variables, and Fisher’s exact test for dichotomous indicator (0–1) variables. Comparisons between the time trajectories (from baseline to two years) for LVEF, infarct zone wall motion, border zone wall motion, infarct size, LV mass, and LV end diastolic volume and LV end systolic volume indices (LVEDVI, LVESVI) were carried out using a repeated measure general linear model. These models included terms for trajectories, treatment effect (BMC versus placebo), and trajectory-treatment interactions. In order to assess the ability of MVO to explain differences in LVEF from baseline to 6 months, a multiple regression model was carried out. The change in LVEF was the dependent variable and baseline MVO was the explainer variable of interest. Additional explainer variables included gender, baseline LVEF, baseline LVEDVI, baseline LVESVI, and baseline infarct size. Clinical events were evaluated using a chi-square test for proportion. Any p-value less than 0.05 was considered statistically significant with no adjustment for multiplicity.

RESULTS

Cohort sizes for cMRI data

Between July 2008 and November 2011, 120 patients were enrolled and randomized (BMC=79, placebo=41) of which cMRI analysis was completed in 110 patients at 6-months. Expanding out to 2 years, 85 patients completed baseline, 6 month, 1 year and 2 year cMRI; these patients comprise the 2-year cohort (BMC=58, placebo=27) (Figure 1).¹⁰ This 2-year cohort represents 71% of all randomized participants (n= 85/120). Loss of analyzable MRIs over 2-years was due to the following reasons: ICD implants (n=10), death (n=3), lost to follow-up (n=7) and refused or other MRI contraindication (n=15).

Figure 1.

Patient flowchart for TIME main trial and MVO substudy.

Baseline characteristics

Baseline characteristics of the 2-year cohort appear in Table 1. Cardiac risk factors were uniformly distributed between the two groups with the exception of hypertension history that was more prevalent in the placebo group (p=0.002).

Global and regional LV function

Overall, for the 2-year cohort there was a non-significant absolute increase in global LVEF in both groups: from 45.9 ± 9.4 to 48.7 ± 11.2% in the BMC group and from 46.9 ± 8.7 to 51.6 ± 11.7% in the placebo group from baseline to 2 years (Table 2).

Table 2.

MRI Analysis for TIME 2-Year Cohort

	BMC Change from 3 Day						Placebo Change from 3 Day
	N	Mean	SD	Mean	SD	P-value*	N	Mean	SD	Mean	SD	P-value*	P-value†
LVEF
Baseline	58	45.9	9.4				27	46.9	8.7
6 month	58	50.3	11.1	4.4	9.4		27	51.6	11.2	4.7	11.8
1 year	58	49.8	11.8	3.9	9.2		27	50.0	10.8	3.2	11.4
2 years	58	48.7	11.2	2.8	9.4	0.381	27	51.6	11.7	4.7	12.0	0.324	0.302
Wall Motion IZ (mm)
Baseline	58	3.9	4.8				27	4.7	4.7
6 month	58	6.1	6.6	2.2	6.0		27	8.2	6.0	3.6	5.5
1 year	58	6.5	5.8	2.6	4.7		27	6.6	5.4	2.0	6.1
2 years	58	6.0	6.3	2.1	5.4	<.0001	27	7.0	6.0	2.4	6.6	0.009	0.175
Wall Motion BZ (mm)
Baseline	58	16.3	10.4				27	14.9	10.1
6 month	58	20.7	11.2	4.4	7.6		27	21.6	13.2	6.7	10.2
1 year	58	21.7	11.5	5.3	7.3		27	22.1	13.0	7.2	11.1
2 years	58	21.3	12.1	5.0	7.6	<.0001	27	20.4	13.3	5.5	10.8	<.0001	0.394
Infarct Size (g)
Baseline	56	44.1	23.1					27	48.4	27.9
6 month	56	29.8	15.1	−14.2	17.5			27	31.5	20.9	−16.9	24.0
1 year	56	28.2	15.3	−15.8	16.9			27	28.1	17.9	−20.4	26.6
2 years	56	25.0	14.4	−19.1	15.7	<.0001	27	22.6	12.1	−25.9	21.6	<.0001	0.042
LV Mass (g)
Baseline	56	179.2	48.8				27	180.4	47.1
6 month	56	155.1	42.2	−24.1	23.0		27	163.6	43.9	−16.8	27.0
1 year	56	148.1	43.8	−31.0	21.8		27	153.6	45.2	−26.8	24.2
2 years	56	143.0	38.2	−36.2	25.4	0.001	27	149.9	44.1	−30.5	21.5	0.092	0.500
LVEDVI (ml/m2)
Baseline	58	76.7	17.7					27	69.4	17.6
6 month	58	87.7	25.2	11.0	15.5			27	77.5	22.0	8.2	18.3
1 year	58	90.1	24.6	13.4	16.0			27	80.9	21.9	11.5	18.4
2 years	58	85.2	25.3	8.5	20.0	0.050	27	78.2	22.7	8.8	18.6	0.272	0.652
LVESVI (ml/m2)
Baseline	58	42.0	13.7				27	37.0	11.8
6 month	58	45.0	20.5	3.0	12.8		27	38.6	17.3	1.6	15.2
1 year	58	46.9	21.7	4.9	13.6		27	41.7	18.1	4.7	15.2
2 years	58	45.3	21.6	3.3	15.5	0.302	27	38.6	16.6	1.5	12.7	0.969	0.696

^*P-value for trend;

^†P-value for treatment effect

BMC=bone marrow cells; SD=standard deviation; LVEF=left ventricular ejection fraction; IZ=infarct zone; BZ=border zone; LVEDVI=left ventricular end diastolic volume index; LVESVI=left ventricular end systolic volume index

In the 2-year cohort, mean wall motion in the infarct zone increased in both groups: from 3.9 ± 4.8 to 6.0 ± 6.3 mm in the BMC group (p<0.001) and from 4.7 ± 4.7 to 7.0 ± 6.0 mm in the placebo group (p=0.009) (Table 2). Similarly, in the infarct border zone, mean wall motion increased from 16.3 ± 10.4 to 21.3 ± 12.1 mm in the BMC group (p<0.001) and from 14.9 ± 10.1 to 20.4 ± 13.3 mm in the placebo group (p<0.001). The greatest increase in LV function occurred between baseline and 6-months with minimal change between 6-months and 2-years.

Infarct size and LV mass

In the 2-year cohort, from baseline to 2-years, infarct size significantly decreased in both groups: from 44.1 ± 23.1 to 25.0 ± 14.4 g in the BMC group and from 48.4 ± 27.9 to 22.6 ± 12.1 g in the placebo group; both p<0.001. Infarct size declined at a greater rate in the placebo group than the treatment group (p=0.042). Additionally, LV mass decreased in both groups: from 179.2 ± 48.8 to 143.0 ± 38.2 g in the BMC group (p=0.001) and from 180.4 ± 47.1 g to 149.9 ± 44.1 g in the placebo group (p=0.092) (Table 2).

Left-ventricular volumes

In the in the 2-year cohort, left-ventricular end-diastolic volume index (LVEDVI) and end-systolic volume index (LVESVI) increased from baseline to 1-year and then stabilized through 2-years in both groups. In the BMC group, over 2 years LVEDVI increased from 76.7 ± 17.7 to 85.2 ± 25.3 ml/m² (p=0.050) and from 69.4 ± 17.6 to 78.2 ± 22.7 ml/m² in the placebo group (p=0.272); while LVESVI had very little change in either group (Table 2).

Cohort sizes for cMRI and Microvascular Obstruction (MVO)

At baseline, there were 115 patients who had analyzable MVO data, however, of these, only 108 patients also had 6 month cMRI data. Thus, this MVO analysis was conducted solely in patients with 6 month follow-up cMRI data. Of these 108 patients, 60 patients had no MVO on their baseline cMRI scans, while 48 patients or 44% had MVO present, a finding similar to that observed in a recent MVO meta-analysis of STEMI patients treated with primary PCI.¹¹

MVO findings

Patients with MVO tended to have lower baseline LVEF (MVO present=42.8 ± 10.3 vs. MVO absent=46.5 ± 9.8%; p=0.057) and greater LVEDVI (MVO present=80.7 ± 18.4 vs. MVO absent=71.2 ± 16.4 ml/m²; p=0.007) and LVESVI volumes (MVO present=46.1 ± 13.2 vs. MVO absent=38.5 ± 12.5 ml/m²; p=0.003) and infarct size (MVO present=56.5 ± 27.9 vs. MVO absent=36.2 ± 19.0 g; p<0.001) (Online Table I). There was also a marked disparity in the sex of patients with MVO. Only 1/11 women had MVO vs. 47/97 men. MVO was independently associated (p=0.003) with the change in LVEF from baseline to 6 months, after adjusting in a multiple regression model for the simultaneous influences of gender (p=0.350), baseline LVEF (p=0.022), LV end diastolic volume index (p=0.184), LV end systolic volume index (p=0.086) and infarct size (p=0.279).

Patients with MVO at baseline (n=48) experienced an increase in LVEF at 6-months of only 0.2 versus 6.2 among those without MVO (n=60) (p=0.003). In addition, patients with MVO presence was associated with significantly greater adverse LV remodeling at 6-months vs. patients without MVO (Figure 2).

Figure 2. Change in LV function between baseline and 6-months stratified by presence or absence of MVO at baseline.

A) The presence of MVO was associated with little improvement in LVEF at 6 months (0.2 absolute EF units) compared to those patients without MVO (6.2 absolute EF units). B) Change in LVEDVI and LVESVI showed patients with MVO had much greater adverse LV remodeling compared to patients without MVO at 6 months.

In the 2-year cohort (patients with cMRI at baseline, 6 months, 1 year and 2 years), 83 patients had analyzable MVO at baseline. At 2-years, patients with MVO (n=35) continued to have reduced LVEF and greater LV volumes and infarct size compared to those patients without MVO (n=48) (Table 3). However, the disparity between the groups lessened as patients with larger infarcts had more associated severe LV dysfunction and were not included due to ICD implantation (Figure 3). To our knowledge, this represents the longest cMRI imaging follow-up of patients with MVO in the literature.

Table 3.

MRI analysis of TIME 2-Year Cohort Stratified by Presence or Absence of MVO at Baseline

	MVO Present			MVO Absent			Mean Difference	SE	P-value T-test
	N	Mean	SD	N	Mean	SD
LVEF to 6 months
Baseline	35	44.9	7.8	48	47.3	10.1
6 months	35	46.7	9.9	48	54.3	10.6
Change	35	1.8	9.7	48	7.0	9.5	5.2	9.6	0.018
LVEF to 2 year
Baseline	35	44.9	7.8	48	47.3	10.1
2 year	35	46.7	10.4	48	52.1	11.7
Change	35	1.8	10.7	48	4.8	10.0	3.0	10.3	0.192
LVEDVI to 6 months
Baseline	35	80.1	16.8	48	70.2	17.9
6 months	35	95.8	23.3	48	76.8	22.8
Change	35	15.8	15.4	48	6.6	16.0	−9.1	15.8	0.011
LVEDVI to 2 year
Baseline	35	80.1	16.8	48	70.2	17.9
2 year	35	91.8	24.8	48	77.5	22.8
Change	35	11.7	19.9	48	7.3	18.8	−4.5	19.3	0.305
LVESVI to 6 months
Baseline	35	44.3	12.4	48	37.5	13.4
6 months	35	52.3	20.3	48	36.1	16.6
Change	35	8.0	13.9	48	−1.4	11.8	−9.4	12.8	0.002
LVESVI to 2 year
Baseline	35	44.3	12.4	48	37.5	13.4
2 year	35	49.6	18.5	48	38.7	20.7
Change	35	5.3	12.3	48	1.2	16.2	−4.1	14.7	0.191
Infarct Size to 6 months
Baseline	33	57.4	27.8	48	37.3	19.2
6 months	33	38.1	18.2	48	25.2	14.6
Change	33	−19.3	21.3	48	−12.1	18.7	7.2	19.8	0.121
Infarct Size to 2 year
Baseline	33	57.4	27.8	48	37.3	19.2
2 year	33	30.8	13.6	48	19.6	12.2
Change	33	−26.6	19.7	48	−17.7	16.3	8.9	17.8	0.037

SE=standard error; LVEF=left ventricular ejection fraction; LVEDVI=left ventricular end diastolic volume index; LVESVI=left ventricular end systolic volume index

Figure 3. Effect of Lost to Follow-up on Long-Term Evaluation of LV Function.

A) Change in LVEF over 2 years in the 2-year cohort. B) Change in LVEF between baseline and 6 months in those patients who completed only the 6 month cMRI follow-up and then were lost to follow-up due to death, ICD implantation or MRI contraindication.

Major Adverse Cardiac Events (MACE)

Looking at the TIME main trial cohort, at 2-years, a total of 21 patients in the BMC group experienced 28 major adverse cardiac events (protocol-designated MACE) while 10 patients in the placebo group had 18 major adverse cardiac events (p=NS; Online Table II). There were three deaths in the BMC group and none in the placebo group; one death due to motor vehicle accident, another due to intracerebral bleed from an intracranial aneurysm prior to stem cell infusion and a presumed sudden cardiac death in a third patient who was found unresponsive in a parking lot. There were 17 repeat revascularizations (BMC=11; placebo=6) and seven hospitalizations for heart failure (BMC=5; placebo=2). Notably, of the 10 patients receiving ICDs, eight had underlying MVO (Online Table III).

DISCUSSION

TIME was the first cell therapy trial sufficiently powered to examine the effect of timing of cell delivery on the recovery of LV function and the first trial to administer the same dose of cells in a network setting using a local, automated cell processing device.⁸ TIME⁶ was developed in parallel with LateTIME⁷, which investigated if delayed delivery of cell therapy (2–3 weeks) in a similar STEMI population would also enhance the recovery of LV function. However, both trials individually and combined (Δ LVEF = −1.4 ± 9.5 %; p=0.967)¹² were notable for the failure of BMCs to improve LV function when measured at 6-months over the improvement observed in patients receiving cell-free placebo. SWISS-AMI¹³ also independently examined the role of timing of cell delivery in a similar STEMI population. In SWISS-AMI, patients were randomized (1:1:1) to cell delivery on Day 5–7 versus 3–4 weeks compared to a control group. Similar to TIME and LateTIME results, no treatment effect of BMCs on LV functional recovery was detected at 4-months or at one year follow-up by cMRI.¹³ Our 2-year cMRI follow-up of patients with moderate to large anterior STEMIs represents one of the longest serial MRI studies in the literature.

TIME was developed following the findings of REPAIR-AMI² and BOOST³ that demonstrated intracoronary delivery of autologous BMCs improved LV function following STEMI. However, since these publications, the results of BMC trials have been mixed. Although several meta-analyses have found a small treatment effect (3%) of BMCs on the recovery of LV function post-STEMI^{14, 15}, it is notable that this treatment effect seems to disappear when cMRI imaging is utilized^{15, 16} or when individual patient data are analyzed.¹⁷

LV function and volumes

Both global LVEF and regional LV function in the infarct and border zones increased similarly in the BMC and placebo groups from baseline to 6-months and then remained relatively stable out to 2-years of follow-up with no difference between groups. Much of the initial improvement in LV function could be attributed to the resolution of myocardial stunning that occurs days to weeks following reperfusion. In support of this, in LateTIME the increase in LVEF between baseline (2–3 weeks) and 6-months was much less than in TIME due to the stipulated difference in the timing of the baseline LVEF (3.2% vs. 1.4%). These early changes in LV function following reperfusion highlight one of the limitations of using the change in LVEF as a primary endpoint in STEMI trials.

The increase in LVEF observed at 6-months is consistent with previous serial imaging studies of STEMI in many non-cell therapy trials. Ripa et al.¹⁸ performed serial cMRI imaging in 58 patients with STEMI and observed that LVEF increased from 52.9 to 61.0 % at 6-months with the greatest improvement occurring at one month (59.4%). Ndrepepa et al.¹⁹ measured the change in LVEF in 626 patients following primary PCI over 6-months using LV angiography and observed that LVEF increased from 51.6 to 57.4%. Those patients with the most depressed LVEF at baseline had the greatest recovery of LV function. Similar findings were also observed in the REPAIR-AMI trial² and in the combined TIME and LateTIME populations.¹² These modest improvements in LVEF over 6-months in the STEMI patient population could potentially obscure any treatment effect of cell therapy.

Patients in TIME were at risk of developing adverse left-ventricular remodeling and heart failure due to relatively large anterior infarctions.²⁰ However, the changes in LVEDVI and LVESVI were modest over the first year and actually declined slightly between year-1 and year-2. This may have contributed to the relatively low incidence of heart failure in TIME (n=7) despite infarctions that averaged 25% of the LV and supports the benefit of the ongoing medical therapy and close observation that these patients received as part of a clinical trial. These small changes in LV volumes over several years were also observed by the ASTAMI Investigators between 2–3 weeks post-PCI and 3-years by cMRI imaging.²¹

Although cMRI represents an ideal imaging modality for the serial follow-up of infarct size and LV function of STEMI patients in cell therapy trials, it is affected by patient drop-out due to clinical events such as device (ICD or LVAD) implantation, heart transplantation, and death.²² Since this occurs more frequently in patients with more severe LV dysfunction, it effectively acts to mitigate any decline in ejection fraction or adverse LV remodeling of the cohort over time. This may create an impression of overall stability of LV function in the follow-up period. This is reflected in our follow-up data demonstrating that patients who completed only 6-months of MRI follow-up (n=15) due to ICD implantation or other causes had significantly lower LVEFs than the overall cohort at baseline (37.5 vs. 46.1%). Furthermore, this subgroup experienced an overall decline in LVEF at 6 months (−3.1%) compared to the improvement in the remainder of the cohort (+ 4.5%) (Figure 3). This issue may arise more frequently as sicker populations of patients are enrolled in cell therapy trials.

Importantly, the CCTRN has recently developed cMRI protocols to permit imaging of patients with ICDs and pacemakers that should help to reduce losses in image acquisition over time.

Infarct size and MVO

cMRI measurement of infarct size by late gadolinium enhancement (LGE) has higher reproducibility and lower variability compared to SPECT²³ and is a strong predictor of adverse outcomes and mortality.²⁴ Infarct size in TIME patients was large at baseline, averaging 25% of LV mass. However, a marked decline (> 30%) was observed in infarct size over 6-months in both groups (Figure 4) with an ongoing small decline out to 2-years. In concert with the reduction in infarct size, a significant and parallel decrease in LV mass was observed. This reduction reflects the ongoing dynamic changes in infarct size that occur over time after reperfusion related to resorption of myocardial edema, adjacent wall thinning and replacement fibrosis of necrotic myocardium.²⁵ These findings are similar to those of Ganame et al.²⁵ who also noted a 40% decline in infarct size in patients following STEMI when measured by cMRI at 1-week and 4-months and the 14% decline in LV mass over 1 year in the study of O’Regan et al.²⁶ To the best of our knowledge, our 2-year MRI follow-up is unique in the literature for its duration of follow-up documenting this ongoing decline in infarct size and LV mass.

Figure 4. Serial measurement of infarct size (g) by cMRI from baseline to 2 years in the bone marrow cell (BMC) and placebo groups.

Serial measurement of infarct size (g) by cMRI from baseline to 2 years in the BMC and placebo groups.

In the acute period (days), infarct size may increase significantly due to the accumulation of myocardial edema and inflammatory cell infiltration that is accompanied by uptake of gadolinium resulting in an overestimation of irreversible injury. As a result, many LV segments with near transmural LGE can recover contractile function over time as the transmural extent of LGE is reduced.^{27, 28} In support of this concept, recovery of wall motion was observed in many segments in the stipulated infarct zone during the first 6-months of follow-up.

Although T2-imaging was not performed to measure myocardial edema, the baseline MRIs were performed during a period of maximal edema accumulation.²⁹ Over months, the gradual resorption of the infarct and edema contributes to the observed reduction in infarct size. Thus, cell therapy studies utilizing change in infarct size as a primary endpoint must be cognizant of these dynamic changes in infarct size.

Late MVO was observed in approximately 50% of patients. This is in agreement with other cMRI-based studies when measured several days following STEMI.¹¹ The presence of MVO was associated with marked reductions in the recovery of LVEF and larger LV volumes at 6-months, and is consistent with the findings of a recent MRI-based meta-analysis following myocardial infarction.³⁰ Although the clinical consequences of MVO over several years measured by MRI have been well described in the literature, ours is the first study to show the long-term consequences of MVO on LV function and volumes by cMRI.³¹ Presence or absence of MVO was the most powerful determinant of change in LV function at 6 months irrespective of therapy.

A noteworthy finding was the significant sex difference in MVO prevalence observed in <10% of women versus 50% of the men. A similar finding was observed by others where women had significantly less MVO and greater myocardial salvage than men despite similar ischemic times during STEMI.³² The mechanism for this sex-related finding is unknown but it presents an important knowledge gap and potential target for investigation into the cause and prevention of MVO given its adverse effects on LV function demonstrated in this study.

CONCLUSIONS

The initial lack of benefit of BMC administration on the recovery of global and regional LV function observed in patients with moderate to large anterior STEMI’s is maintained when measured at 2-years, regardless of day of cell delivery. Importantly, no concerns associated with the safety of using BMCs were observed in this high-risk cohort who demonstrate ongoing stability of LV function and volumes out to two years. The presence of MVO was observed in almost half the cohort at baseline and was associated with a significant reduction in the recovery of LV function and adverse left ventricular remodeling and increased need for ICD placement.

Novelty and Significance.

What Is Known?

• Microvascular obstruction (MVO) observed on cardiac MRI following ST-elevation myocardial infarction (STEMI) is associated with adverse left-ventricular (LV) remodeling and poor clinical outcomes.

• The long-term effects of MVO on cardiac MRI of LV function and infarct size in response to cell therapy with bone marrow mononuclear cells (BMCs) has not been previously described.

What New Information Does This Article Contribute?

• Cell therapy with BMCs did not improve global or regional LV function compared to placebo in patients with anterior STEMIs and moderate to severe LV dysfunction over 2 years of follow-up.

• The presence of MVO on baseline (Day 3) cardiac MRI was associated with a significant reduction in the recovery of LV function and adverse left ventricular remodeling and increased need for ICD placement at 2-years of follow-up.

• The use of cardiac MRI for endpoint analysis in cell therapy trials is adversely affected by patient drop-out due to clinical events such as device implantation, that occurs more frequently in patients with more severe LV dysfunction.

The TIME Trial was developed by the Cardiovascular Cell Therapy Research Network to investigate the effect of timing (Day 3 vs. Day 7) of BMC delivery on the recovery of global and regional LV function in 120 patients following anterior STEMI. Pre-specified cardiac MRI measurements of LV function were performed at baseline, 6-months, 1 and 2-years. In one of the longest serial cMRI imaging analyses of patients with large anterior STEMIs, BMCs had no effect on the recovery of LV function, volumes or infarct size compared to placebo at any time point over 2 years of follow-up. No effect of timing of BMC administration was observed. MVO was observed in half the cohort on baseline MRI and was associated with reduced recovery of LV function, greater adverse LV remodeling and more ICD implantations. The use of cardiac MRI results in greater patient loss over time due to device implantation and occurred in patients with more severe LV dysfunction resulting in overestimation of clinical stability of the cohort.

Nonstandard Abbreviations and Acronyms

BMC

bone marrow mononuclear cells

CCTRN

Cardiovascular Cell Therapy Research Network

LV

left ventricular

LVEDVI

left ventricular end diastolic volume index

LVEF

left ventricular ejection fraction

LVESVI

left ventricular end systolic volume index

MRI

magnetic resonance imaging

MVO

microvascular obstruction

NHLBI

National Heart, Lung, and Blood Institute

PCI

percutaneous coronary intervention

STEMI

ST-elevation myocardial infarction