Metabolic Mapping and Cardiac Resynchronization (Aim 1)
Phase: NA
Conditions: Heart Failure
Sponsor: Yong-Mei Cha

 
Metabolic Mapping and Cardiac 
Resynchronization (Aim 1) 
 
NCT# 03420833 
 
June 20, 2021 

Protocol version 10 dated June 20, 2021 
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STUDY OVERVIEW 
 
Title  
Metabolic Mapping and Cardiac Resynchronization (Aim 1) 
Running Title Metabolic Mapping and CRT 
IRB Protocol 
Number 
17-003359 
Phase 
Pilot 
Principal 
investigator 
Yong-Mei Cha, MD 
Mayo Clinic 
Objective 
To determine early alterations in metabolic pathways and metabolomics 
biomarkers in patients with mild heart failure (HF) and left bundle branch block 
(LBBB), and their role in the prediction of outcomes by early CRT. 
Study Design Single-center, randomized cross-over study 
Overall Study 
Design 
We plan to complete subject enrollment by the end of Year 3. Data validation, 
adjudication and analysis will be finished by the end of Year 5. 
Subject 
Participation 
Duration 
Approximately 12 months 
Primary 
Objectives 
Primary efficacy objectives (composite endpoint) 
The study primary endpoint is a reduction in the LV end-systolic volume index 
(LVESVI, reverse LV remodeling) in the CRT-ON gorup as compared to the CRT-
OFF group. 
Primary Safety Objective 
• 
Freedom from system-related complications in randomized subjects will 
be calculated at 1, 6, and 12 months post implant to determine the safety 
profile, including the device pocket hematoma, pneumothorax, myocardial 
performation, lead dislodgement, lead revision, and device-related 
infection.. This safety profile will be balanced against efficacy to quantify 
the risk-benefit ratio. 
 
Secondary 
Objectives 
Secondary efficacy objectives 
• 
Hospital admission for HF 
• 
Increase in NT pro-BNP level by 30% 
• 
Sustained ventricular tachycardia or fibrillation greater than 30 seconds 
• 
Decrease in LVEF to ≤34% 
• 
Death 
 
Device 
BIOTRONIK Edora HF-T QP, or Ilivia HF-T QP DF4 
Number of 
Subjects 
100 subjects (of these 30 will be female) 
Subject 
Inclusion 
Criteria 
• 
LVEF 35%-50% 
• 
NYHA class I-II  
• 
QRS duration of ≥120ms 
• 
Left bundle branch block (LBBB) 
• 
Patient is able to receive a transvenous pectoral CRT implant 

Protocol version 10 dated June 20, 2021 
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• 
Patient is able to sign informed consent 
• 
Two echocardiograms are required to confirm LVEF≤ 50% (one echo 
must be within 12 months of enrollment) 
• 
Patient is on optimal and stable medical therapy (ACE inhibitor or AT1 
blocker, beta blocker, etc. over the last 6 months) 
 
Subject 
Exclusion 
Criteria 
• 
Advanced comorbid conditions with life expectancy <1 year 
• 
Patient is <18 of years of age 
• 
Patient has a CRT device 
• 
Female patients who is pregnant or not on a reliable form of birth control. 
Women of childbearing potential are required to have negative pregnancy 
test within the 7 days prior to device implant 
• 
Unwilling or unable to return for required follow-up visits 
• 
Patient decides study participation is cost-prohibited 
 
Duration 
of 
Exposure 
At the 12-month follow-up, when each subject completes his/her cross-
over 6-month therapy, the CRT (Edora HF-T QP, or Ilivia HF-T QP DF4) 
function will be programmed on to provide chronic benefit from CRT for a 
battery life of 8.6 years. 
Reference 
therapy 
Patients are self-referenced as a cross-over design. 
Statistical 
Methodology 
The primary endpoint is the change in LVESVI.  We will compare the changes in 
LVESVI between CRT-ON and CRT-OFF groups. 
We will explore 5-10 metabolomic factors as principal components to 
predict group differences (using a 2-sample t-test), and response to CRT 
therapy. In particular, we will relate these principal components to 
response to CRT using multiple linear regression (for the LVESVI 
endpoint) and Cox Regression (for the Clinical endpoint).   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol version 10 dated June 20, 2021 
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RESEARCH STRATEGY 
A. Background and Significance 
Heart failure is a progressive disease with high mortality and morbidity. Heart failure (HF) affects 5.7 
million American adults and claims nearly 300,000 lives annually.1 HF progresses along a continuum from 
asymptomatic (Stage A and B) to symptomatic (Stage C and D).2 Nearly three times as many people with 
left ventricular (LV) systolic dysfunction are in Stage B as in Stages C and D combined.3,4 In the 
Framingham Heart Study, total mortality rates within four years of symptomatic HF development were 24% 
in women and 55% in men.5 In the community, the prevalence was 6% for those with a left ventricular 
ejection fraction (LVEF) of 50% or less and 2% for those with an LVEF of 40% or less.6 This translates to 
4% of the population who have mild LV systolic dysfunction with LVEF between 41%-50%.7 Although the 
risk of hospitalization or death declines as LVEF increases, an LVEF in the range of 36% to 50% still 
confers a considerable risk of adverse outcomes. For instance, the CHARM study showed that patients with 
an LVEF between 33%-52% had annual cardiovascular mortality rates in the range of 4.0%-6.0% and HF 
hospitalization rates of 5.7%-7.2%.8  
Left bundle branch block (LBBB) and ventricular dyssynchrony. LBBB is frequently (>20%) 
accompanied by progression of LV systolic dysfunction, with the consequence of delayed electrical 
conduction to the LV lateral wall.9,10 This recognized ventricular dyssynchrony further reduces myocardial 
efficiency and cardiac output, worsening cardiac performance. Compared with normal ventricular 
conduction, the presence of LBBB is associated with a near two-fold increase in all-cause mortality.11 
Cardiac resynchronization therapy (CRT) resynchronizes electrical and mechanical coupling by 
simultaneously exciting the right and left ventricles. It improves chronic LV systolic dysfunction and reverse 
LV remodeling in patients with LVEF of 35% or lower.12-14 The benefit of CRT is more favorable in patients 
with a widened QRS complex and LBBB.12,14-23 Yet, the metabolomics signature of LBBB and metabolic 
pathways affected have not been well determined. Based on our own preliminary data (see below), we 
propose that early CRT, in patients with LVEF of 35%-50% and LBBB, may have potential clinical impact 
that reverses the initial stages of LV metabolic and structural remodeling and prevents HF progression to an 
advanced stage.24 
Metabolomics and biomarkers in HF and CRT. HF is characterized by perturbations in the metabolic 
processes including impaired oxidative phosphorylation and compromised ATP production.25-33 Cardiac 
remodeling in HF involves ventricular electrical and mechanical dyssynchrony, which results in metabolic 
heterogeneity and energy insufficiency.23,25-27,34 Heterogeneous myocardial oxidative metabolism has been 
characterized, especially in patients with ischemic cardiomyophaty and LBBB.35 As such, metabolomics 
profiling, when used in combination with standard HF biomarkers (e.g., B-type natriuretic peptide [BNP]), 
has significant diagnositic and prognostic values, and facilitates understanding the mechanisms of 
metabolic derrangements and assessing therapeutic outcomes.36-52 One of the mechanistic benefits of CRT 
is to reduce mechanical dyssynchrony within the left ventricle with subsequent improvement in LV efficiency 
and oxygen consumption.36 However, how CRT affects the myocardial energy and substrate metabolism, 
and which metabolic state and pathways facilitate myocardial recovery, are unknown. Metabolomic analysis 
demonstrates increasing potential to provide individualized predictive information for disease progression 
and precision of treatment.32,37-45,49-52 Determining the associations between metabolomic changes and CRT 
is of importance to understand how this therapy improves cardiac metabolism.53-55 Our preliminary 
investigations have discovered an energetic and metabolomic signature in HF, and potential therapeutic 
effects of CRT on the metabolomic profile.28,52,56 Thus, assessment of the metabolomic state before CRT 
may provide new biomarkers for evaluation of treatment efficacy from CRT.45,51  
Gender difference in CRT outcome. Although 50% of patients with HF are women, they constitute less 
than 25% of the patients enrolled in CRT trials. Despite their underrepresentation in clinical trials, women 
may benefit more than men from CRT.18,19,57-59 The more favorable benefit of CRT for women may be 
associated with differences in baseline disease characteristics. A greater proportion of women have 
favorable factors such as a substrate of nonischemic cardiomyopathy (NICM) and a LBBB pattern, whereas 

Protocol version 10 dated June 20, 2021 
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Figure 1: Clinical database screening process for 
patients with LVEF 36%-50% and LBBB, and matched-
control group.  
a greater proportion of men have ischemic cardiomyopathy (ICM) and renal dysfunction, which are 
associated with a poor prognosis. In the proposed study, we will particularly enroll a higher proportion of 
female  patients  than  the clinical trials to address whether sex is  a biological variable in CRT outcome. We 
aim to determine the cardiac metabolomic signature and active metabolic pathways of women, which may 
differ from that of men, as an underlying mechanism by which women have a more favorable outcome.45,60  
B. Innovation 
Discovery of novel metabolomic biomarkers and transcardiac metabolic signature. Application of 
unbiased metabolomics and new stable isotope technologies to address metabolomic dysregulation in HF 
and metabolic reprogramming by effective CRT is an innovative approach, which will permit underpinning 
metabolic pathways and mechanisms that allow to improve CRT efficacy. This translational study will 
combine cutting-edge metabolomic technologies available at Mayo Clinic to allow, for the first time, the 
determination of whole blood metabolite dynamics using 18O stable isotopes along with plasma and 
transcardiac metabolomic signatures and substrate utilization patterns to uncover the mechanistic link in HF 
and CRT. These measurements will permit mapping cardiac metabolic heterogenity and the homogenizing 
effects of CRT critical for assessing treatment efficacy.35 On the basis of preliminary data, the array of 
metabolomic data collected will reflect the patient’s HF and therapeutic metabolic status, which is an 
integral outcome of the upstream genomic, transcriptomic, and proteomic events, and will allow the 
identification of the exact phenotype, potential biomarkers, and mechanisms of underlying diseases.  
Discovery of cardiac metabolomics pathways that facilitate favorable CRT outcomes in women. We 
will evaluate the metabolic reserve, which is the capacity to increase metabolism of energy providing 
substrates in response to an increase in the workload of the heart.61 Here the innovation lays in discovering 
a potential gender difference in metabolic traits and its mechanistic association with CRT outcomes through 
metabolic pathways using advanced metabolomic technologies. Specific focus will be paid to transcardiac 
metabolite differences and whole blood phosphometabolite turnover and dynamics of substrate shuttles and 
pathways, such as acyl-carnitine and branched-chain amino acid metabolism to detect subtle changes in 
metabolic state. 
Effective treatment of HF in the early stage by CRT. This study proposes to investigate the novel use of 
CRT in patients who have mild HF, with a decreased LVEF of 35% to 50% and a LBBB. This patient 
population is at high risk for progression from mild to severe HF, which represents an increasing burden to 
the community and the health care system. Reversal of myocardial dysfunction in the early stage, before it 
becomes irreversible, is a sound and innovative strategy. 
C. Approach 
Preliminary results 
Mild HF and clinical outcomes  
We screened the Mayo Clinic electrocardiography (ECG) 
database for patients with a diagnosis of LBBB from 1994 
through 2014. Among 12,879 patients identified, 1,436 
patients 
met 
the 
criteria 
of 
having 
transthoracic 
echocardiography (TTE) within one-year of the ECG 
diagnosis of LBBB and a LVEF of 36% to 50% (Figure 1). 
These study patients were matched 1:1 to 1,436 patients 
without LBBB on the basis of age, sex, baseline LVEF, and 
year of TTE. The mean (SD) age in both groups was 67 
(13) years, and 54% were men. Clinical outcomes were 
compared between the LBBB and control groups. 
Long-term survival. The Kaplan-Meier 20-year probability 
of survival in both groups is shown in Figure 2. Patients 
with mild HF (LVEF 36%-50%) and LBBB had lower 
survival rates at 5 (72% vs. 76%; P=.04), 10 (53% vs 61%; 
P<.001), and 20 years (27% vs 46%; P<.001) compared 
with the controls who had a normal QRS duration. 

Protocol version 10 dated June 20, 2021 
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Figure 2. Kaplan-Meier Survival. Overall 20-year survival in 
patients with LBBB and controls. LBBB, left branch bundle block 
Figure 3. Kaplan-Meier Survival in patients with mild HF and 
LBBB according to LVEF changes at follow-up.  
 
LVEF deterioration to <35%. The event-free rates of 
the LVEF reduction to <35% at 1 year (83% vs 89%) 
and 5 years (63% vs 70%) (hazard ratio, 1.34; 95% 
CI, 1.09-1.63; P=.005—were lower in the LBBB group 
than the control group. This finding suggests that, 
compared with a normal QRS duration, the presence 
of LBBB in patients with mild HF led to a greater 
LVEF deterioration with time, which could be 
attributed to ventricular dyssynchrony. 
Implantable cardioverter-defibrillator and CRT- 
defibrillator implantation. The survival free of an 
implantable cardioverter-defibrillator (ICD) or CRT-
defibrillator (CRT-D) placement in the LBBB group 
was lower than the control group (87% vs 92% at 1 
year; 80% vs 88% at 5 years) (hazard ratio, 1.73; 
95% CI, 1.30-2.30; P<.001). Thus, patients in the 
LBBB group received ICD/CRT-D more often than the non-LBBB control group. This finding is consistent 
with evidence of greater LVEF deterioration in the LBBB group. 
Progression of LV dysfunction in mild HF with 
LBBB. Among the 1,436 patients with LVEF of 36%-
50% and LBBB, 485 had follow-up echocardiography 
within the first two-years. The mean (SD) patient age 
was 65 (14) years, 57% were men, and mean LVEF 
was 42% (13%). Of these 485 patients, 281 (58%) 
had no decrease in LVEF, 103 (21%) had an LVEF 
decrease of 1% to 5%, 43 (9%) had a decrease of 6% 
to 10%, and 58 (12%) had a decrease of more than 
10% (P<.001). Overall, 42% of participants had a 
further LVEF reduction within the next two-years.The 
Kaplan-Meier curves showed a significant difference 
in survival by the extend of  LVEF decrease (P<.001; 
Figure 3). Patients with a decrease of >10% had the 
worst survival.  
Metabolomic profiles in HF and CRT 
Baseline transcardiac metabolomics and acute 
response to CRT. We studied the metabolomic profiles of 18 patients with advanced HF who underwent 
CRT-D implantation and 7 controls who underwent catheter ablation for supraventricular tachycardia. Blood 
samples were collected from the peripheral 
vein, femoral artery, and coronary sinus at 
baseline. Metabolomic profiling of plasma 
samples 
was 
performed 
using 
gas 
chromatography-mass 
spectrometry 
(GC-
MS) 
and 
nuclear 
magnetic 
resonance 
spectroscopy 
(NMR).52 
Compared 
with 
controls, the HF patients had an increased 
release of myocardial succinate, which is a 
biomarker of mitochondrial dysfunction, and 
increased uptake of fatty acids and ketone 
bodies 
(2-HB) 
indicating 
alterations 
in 
substrate 
metabolism 
and/or 
deposition 
(Figure 4). After acute biventricular pacing 
for 15 minutes, the repeat transcardiac 
Figure 4. Transcardiac metabolomics changes (peripheral vs coronary 
sinus) in HF patients (blue) compared with controls (red). 2-HD -2-
hydroxybutyrate; * P<.05, **P<.01. HF, heart failure 

Protocol version 10 dated June 20, 2021 
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Figure 6. Transitions in metabolomics states from control to mild heart 
failure (HF mild), to severe heart failure (HF severe) and 3-month after 
CRT in severe heart failure (HF-CRT). Principal component (OPLS-DA) 
analysis of metabolomics profiles. 
metabolomic profile shifted to a different 
state, with increased myocardial uptake and 
metabolism of free fatty acids and decreased 
release of creatinine.  
Metabolomic profile in severe HF and 
chronic 
response 
to 
CRT. 
In 
our 
prospective study of the effect of chronic  
CRT  on  plasma  metabolomic profiles,52 we  
analyzed 24  patients who had  CRT 
implantation  for 
advanced HF (LVEF≤35%) and 10 controls 
who 
underwent 
catheter 
ablation 
for 
supraventricular tachycardia. Blood samples 
were collected before and 3 months after the 
procedure. We performed a metabolomic 
profiling, using GC-MS and NMR. The 
plasma 
metabolomic 
profiles 
changed 
significantly 3 months after CRT (Figure 5A). 
For each patient with HF, scores of the 
integral 
metabolomic 
profile 
after 
CRT 
clustered separately from the original state 
on the orthogonal partial least squares–
discriminant analysis (PLS-DA) plot, which 
indicates a transition to a new metabolic state. An integrated panel of the most important metabolites for 
discrimination of the CRT group included isoleucine, glutamine, and glycerol-1-phosphate (Figure 5B). The 
pattern of metabolite changes (decrease in succinate and increase in glutamate) indicated an improved 
mitochondrial substrate metabolism after CRT (Figure 5C). Among the most improved metabolic pathways 
were amino acid and lipid metabolism (Figure 5D). 
Metabolomic profile in mild HF. We 
prospectively studied metabolomic profiles 
in subjects with a normal LVEF of >55% 
and LBBB (control, n=9), in patients with 
LVEF of 36%-50% and LBBB (HF mild, 
n=8), LVEF of ≤35% and LBBB before 
(HF severe, n=9) and after CRT (HF-
CRT). Figure 6 illustrates the power of 
metabolomics in different HF stages 
separating from the control group. The 
most 
important 
metabolites 
in 
the 
discrimination of HF severity were amino 
acids (asparagine, lysine, glutamine), 
branched-chain amino acids (isoleucine, 
leucine, 
valine) 
and 
products 
of 
catabolism, 
carnitine 
and 
nucleotide 
degradation. The finding suggests an 
early alteration and progression in the 
amino acid, fatty acid and nucleotide 
metabolism in mild HF with LBBB in 
parallel to the advancement of HF.  
 
 
 
Figure 5. Metabolomic signature of heart failure (HF). (A) Changes in 
plasma metabolomic profile of HF patients and controls by orthogonal 
partial least squares discriminant analysis (OPLS-DA). CRT, cardiac 
resynchronization therapy. (B) Most important metabolites in the 
metabolomic signature of HF by variable importance in the projection 
(VIP). (C) Pattern of plasma metabolite changes in HF patients (n = 24; 
*P < .05; **P < .001). (D) Metabolic pathways most affected in HF as 
deduced from an altered plasma metabolomic profile. 

Protocol version 10 dated June 20, 2021 
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Figure 9. Different Metabolomic Profiles in Patients With Ischemic and 
Nonischemic HF Before and After CRT by orthogonal PLS-DA.  
Figure 8. Patients with LBBB (Control LBBB) have 
different metabolic state which is further exacerbated by 
mild heart failure (HF mild LBBB) as compare to control 
with no LBBB (Control). Principal component (OPLS-DA) 
analysis of metabolomics profiles. n=8-14.
 
To 
further 
specify 
the 
metabolite 
biomarker 
changes, 
we 
measured 
the 
carnitine 
and 
acylcarnitine levels using 
liquid 
chromatography-
tandem 
mass 
spectrometry (LCMS), and 
discovered 
a 
significant 
elevation 
of 
plasma 
carnitine, 
acetylcarnitine 
and 
long-chain 
oleoylcarnitine in mild HF patients compared to controls (Figure 7). Such changes indicate that impaired 
tissue free fatty acid oxidation in mitochondria is associated with an increased release of metabolites into 
plasma and deficient overall lipid metabolism.44  
Metabolomic alteration in LBBB. To further 
understand whether LBBB itself impacts the 
metabolomics profile, we compared metabolic 
measurements in subjects, who had an ECG 
diagnosis of LBBB with a normal LVEF of >55%, to 
the control group who had a normal QRS and LVEF, 
and the group who had mild HF (LVEF 36%-50%) 
and LBBB. Notably, the metabolomics profile in 
LBBB with normal LV function (Figure 8) is different 
from the normal QRS controls and is further shifted 
in patients with mild HF and LBBB. The principal 
components for group separation are lactate, 
glucose, 
carnitine, 
glutamine 
and 
succinate, 
indicating altered pathways of carbohydrate, amino 
acid and nucleotide metabolism by LBBB alone 
without the presence of HF. 
Metabolomic 
profiles 
in 
ischemic 
and 
nonischemic 
cardiomyopathy. In our previous 
study, patients with NICM (n=14) 
and ICM (n=10) had different basal 
plasma 
metabolomics 
profiles 
(Figure 
9). 
After 
CRT, 
metabolomic 
profiles 
shifted 
differently, with NICM showing 
better improvement in the plasma 
metabolomics. 
Compared 
with 
ICM, patients with NICM, had 
higher levels of amino acids, 
lactate, and acetate after CRT 
associated with more active amino 
acid, carbohydrate and fatty acid metabolism. 
 
 
Figure 7. Changes in plasma carnitine and acylcarnitine levels in mild HF. n=7 in each 
group.

Protocol version 10 dated June 20, 2021 
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Figure 10. Metabolomic profiles of responders and nonresponders to cardiac 
resynchronization therapy (CRT). (A) Distinction of basal plasma metabolomic profiles of 
responders and nonresponders to CRT by orthogonal partial least squares discriminant 
analysis (OPLS-DA). (B) An integral panel of the most important metabolites permitting 
prediction of responders by variable importance in the projection (VIP). (C) A panel of 
the most important metabolite differences between responders and nonresponders (*P < 
.05). 
 
Figure 11. Plasma metabolomics profiles of male and female HF patients 
(A); Metabolite differences in female and male HF patients (B). [n = 31 
(male) and 7 (female); *P < .05; **P < .001]. 
Metabolomic profile predicts 
improvement in LVEF after 
CRT. Of the 24 patients with HF 
who received CRT, 10 (41%) 
had an improvement in LVEF by 
>5% and a decrease of 1 or 
greater in the New York Heart 
Association (NYHA) functional 
class; they were considered to 
be CRT responders. This group 
of patients differed in their pre-
CRT 
baseline 
plasma 
metabolomic profile from non-
responders in the orthogonal 
PLS-DA plot analysis (Figure 
10A). The panel of the most 
important metabolites allowing 
distinction between responders 
and non-responders to CRT is 
shown (Figure 10B). Patients 
who responded to CRT had 
higher levels of branched-chain 
amino acids (isoleucine, leucine, 
and valine), essential for protein 
synthesis 
and 
metabolic 
signaling. (Figure 10C; P<.05). It 
indicates branched-chain amino 
acids 
are 
catabolized 
to 
metabolic 
intermediates 
as 
important substrates for energy 
production in the failing heart. 
CRT and gender difference 
Women have a greater response to CRT than men. We conducted a retrospective study of 728 
consecutive CRT recipients at our institution who met the guidelines for CRT-D device placement. The 
outcomes of CRT were compared between women (n=166, 22.8%) and men (n=562, 77.2%). Women were 
found to more often have NICM (68% vs 36%, P<.01). After CRT, as compared to men, women had a 
greater improvement in the NYHA class and a trend greater improvement in LVEF and reverse LV 
remodeling.62 
Women and men with HF have different 
metabolomic profiles. The metabolomic 
profile was prospectively studied in 38 
patients 
(7 
women, 
18%) 
who 
had 
advanced HF with LVEF ≤35%. Male and 
female patients had different plasma 
metabolomic 
profiles 
(Figure 
11A). 
Women had higher plasma levels of 
linoleic acid, palmitoleic acid, glycolic acid 
and glycine, which indicated a better lipid 
and amino acid metabolism than men 
(Figure 11B). A distinct feature of the 
female 
metabolome 
was 
a 
higher 

Protocol version 10 dated June 20, 2021 
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glycerol/glycerol-1-phosphate ratio (mean [SD], 2.79 [1.30] than men, 1.19 [0.18]; P<.05) indicating lower 
triglyceride biosynthesis in women. The NMR data indicates that male metabolome had higher levels of N-
methylhistidine and creatinine, which is associated with higher protein degradation and energetic stress.  
Experimental protocol 
Aim: To determine early alterations in metabolic pathways and metabolomic biomarkers in patients 
with mild HF and LBBB, and their role in the prediction of outcomes by early CRT.  
Rationale 
Reversal of myocardial dysfunction in the early stage is of great importance to prevent the progression of 
HF. Here we will investigate the novel use of CRT in patients who have mild HF and LBBB. This patient 
population is at high risk for progression from mild to severe HF. However, the metabolomics signature of 
mild HF with LBBB and metabolic pathways affected have not been determined.  
Study Patients  
Subject inclusion criteria. LVEF 35%-50%, NYHA class I-II, QRS duration of ≥120ms63, LBBB64, and on 
optimal and stable medical therapy (ACE inhibitor or AT1 blocker, beta blocker over the last 6-month), able 
and willing to sign informed consent.  
Subject exclusion criteria. Advanced comorbid conditions with life expectancy <1 year, unwilling or unable 
to return for required follow-up visits, <18 of years of age, existing CRT device, female patients who is 
pregnant or not on a reliable form of birth control. Women of childbearing potential are required to have 
negative pregnancy test within 7days prior to device implantation. 
Our preliminary studies have indicated that among 1,436 patients with LVEF of 36% to 50% and LBBB, 78 
reside in Olmsted and surrounding counties, 256 reside in Minnesota, and 422 reside in nearby states and 
are living candidates for enrollment. In addition, as a referral center, Mayo Clinic is estimated to have 
approximately 80 to 100 new patients eligible for study enrollment each year. 
Study design and randomization 
We propose a single-center, randomized cross-over study design. The study will be approved by the Mayo 
Clinic Institutional Review Board, and a consent form will be signed by each participant. We will enroll 100 
patients at Mayo Clinic, Rochester, Minnesota, who have LVEF of 35% to 50%. Of these patients, 30 will be 
women. Participants will undergo placement of a CRT-pacemaker (CRT-P) after the completion of the 
baseline assessment if all inclusion and none of the exclusion criteria are met. Participants who have an 
ICD in place, or who meet clinical indication for an ICD, will undergo placement of a CRT-defibrillator (CRT-
D). Defibrillation will not be added for patients who do not have an existing ICD system or do not meet 
clinical indications for an ICD. Participants who have a successful CRT implantation will be randomly 
assigned 1:1 to have the CRT programmed on (CRT-ON, treatment group) or off (CRT-OFF, control group) 
within 7 days of completing the implant procedure. Participants will be randomly assigned using an 
allocation method defined by a biostatistician. Investigators managing HF in study participants will be 
blinded to the randomization method to minimize potential bias. A washout period after the device 
implantation will not be used in this study.19 At 6-month follow-up, the CRT function will be turned on in the 
CRT-OFF group and the CRT function will be turned off in the CRT-ON group in a cross-over design. All 
participants will cross-over at the 6-month follow-up visit. At 12-month follow-up, when each patient 
completes his/her cross-over 6-month therapy, the CRT function will be programmed on to provide chronic 
benefit from CRT for a battery life of 8.6 years. The subjects will be blinded from whether biventricular 
pacing is on or off. For participants who receive a CRT-D, the defibrillation function will be programmed on 
for the duration of the study. 
Study device 
A United States Food and Drug Administration (FDA)-approved CRT device will be used for this study. 
Because the clinical application of CRT in patients with an LVEF of 35% to 50% has not been approved by 
the FDA and current AHA/ACC/HRS guidelines,16 we have obtained the approval of an Investigational 
Device Exemption (IDE) from the FDA for this study aim (FDA-approval letter is attached). BIOTRONIK SE 
& Co. KG has agreed to donate the FDA-approved CRT device, models Edora HF-T QP or Ilivia HFT-QP 
DF4, along with Setrox S, Solia S, Plexa S, Plexa DF-1 S, and Sentus QP leads (agreement letter is 
attached) for the study.  

Protocol version 10 dated June 20, 2021 
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Figure 12. Shows coronary sinus blood sampling locations. The 
circles indicate blood exampling from the coronary sinus ostium 
and the proximal lateral vein from the LV lateral wall (the site of 
latest activation) that reflect the local metabolism will be collected 
and compared.  
Preprocedure Evaluation 
Clinical evaluation. Patient baseline information including age, comorbid conditions such as hypertension, 
coronary artery disease, diabetes mellitus, and etiology of cardiomyopathy (ICM vs NICM) will be collected. 
The HF symptoms (measured by Kansas City Cardiomyopathy Questionnaire: KCCQ), physical signs, 
NYHA class, ECG, echocardiography (may include 3D echo images), and 6-minute walk test will be 
assessed. 
12-Lead ECG. The ECG results will be reviewed by electrophysiologists who are blinded to the patients’ 
clinical status. Intrinsic QRS duration and morphology will be measured and validated.  
Six-minute walk test. The 6-minute walk test will be conducted according to a standardized protocol. 
Patients will perform the test using a 100-foot internal hallway,66, 67 and will be asked to walk the hallway at 
their own pace for 6 minutes. The patient will be supervised by a nurse during the test, and blood pressure 
and pulse rate will  be monitored during the study. 
Echocardiography Two-dimensional echocardiography will be performed to assess changes in LV 
dimension, volumes, and LVEF. Volumes will be estimated by averaging those derived from the 2-chamber 
and -chamber views according to the Simpson method, and the LVEF will be calculated in the usual fashion 
(derived from 2-dimensional measurements of 
diastolic and systolic LV dimension or volumetric 
analysis using the method of disks).17,19 Two 
echocardiograms are required to confirm a stable 
reduced LVEF prior to randomization (one echo 
must be within 12 months of enrollment). Three-
dimensional (3D) echo offers a reproducible 
assessment of left ventricular (LV) structure, 
function, 
volumes 
and 
LV 
mechanical 
dyssynchrony. It has been shown that the LV 
mechanical dyssynchrony quantification by 3D 
echo is reproducible and, systolic dyssynchrony 
index (SDI) is an excellent predictor of response 
to CRT. For the purpose of the study patients will 
undergo an additional 3D echocardiographic 
examination. Datasets will be obtaiıned by 
commercially 
available 
echocardiographic 
systems equipped with 3D probes. Full-volume 6-
beat LV 3D echo data sets will be acquired from 
the apical approach during patient breath hold.  
The LV-3D echocardiographic images will be 
exported in VolDICOM format to a separate 
workstation equipped with dedicated vendor-
independent software packages for 3D echo (4D 
LV-Analysis 3.1, TomTec Imaging Systems, 
Unterschleissheim, Germany).  
LV end-diastolic (EDV) and end-systolic volumes (ESV), LV ejection fraction (EF), strain including global 
longitudinal strain (GLS) and global circumferential strain (GCS) and, systolic dyssynchrony index (SDI) will 
be calculated offline.  
 
 
CRT implantation  
The FDA-approved, commercially available CRT devices and leads will be used. The transvenous LV lead 
will be placed into coronary lateral vein to correct delayed lateral wall activation in LBBB, if possible.68,69 
After the blood sample is taken, the LV lead will be advanced to the distal tributary of this vein. A QRS-to–
local ventricular activation time (QRS-LV time) will be measured to assess the conduction delay.70-72 

Pr ot oc ol v ersi o n  1 0  d at e d J u n e 2 0, 2 0 2 1 
P a g e 1 1  of 2 7  
P ati e nt s  will  b e  o b s er v e d  i n  t h e  h o s pit al  f or  2 4  h o ur s,  a n d  d e vi c e s  will  b e  i nt err o g at e d  b ef or e  h o s pit al  
di s mi s s al.  
Bl o o d s a m pl e c oll e cti o n f or m et a b ol o mi c m e a s ur e m e nt  
P eri p h er al v e n o u s bl o o d s a m pl e s will b e c oll e ct e d b ef or e C R T, at 6 -m o nt h a n d 1 2 -m o nt h f oll o w-u p ( eit h er 
C R T -O N f or 6-m o nt h or C R T -O F F  f or 6-m o nt h ). At i m pl a nt, bl o o d s a m pl e s fr o m f e m or al art er y a n d c or o n ar y 
si n u s  will  b e  c oll e ct e d  t o  m e a s ur e  tr a n s c ar di a c  m et a b ol o mi c  gr a di e nt.  M or e  oft e n ,  th e  L V  l e a d  d eli v er  
s h e at h f or c or o n ar y si n u s c a n n ul ati o n i s a bl e t o b e a d v a n c e d i nt o t h e b e gi n ni n g of l at er al v ei n  t o f a cilit at e 
t h e  L V  l e a d  pl a c e m e nt   (7 0 %- 8 0 %),  w h er e  bl o o d  s a m pl e  will  b e  c oll e ct e d  t o  d et er mi n e  t h e  r e gi o n al  
m et a b o li c pr ofil e a s s h o w n i n Fi g ur e 1 2 . T h e r ati o n al f or c oll e cti n g t h e r e gi o n al bl o o d i s t o d et er mi n e 1) 
w h et h er  t h er e  i s  a  diff er e nti al  m et a b oli s m s  b et w e e n  I C M ( di s cr et e  s c ar  v s  n o  s c ar  a c c e s s e d  b y  
e c h o c ar di o gr a p h y) v s NI C M  ( diff u s e d fi br o si s), a n d 2) m et a b oli c f e at ur e at t h e m o st d el a y e d c o n d u cti o n 
ar e a at t h e L V  l at er al w all i n c o pi n g wit h Q R S-L V ti m e.   
M u s c ul ar s a m pl e s c oll e cti o n f or m et a b ol o mi c s m e a s ur e m e nt  
W h e n t h e p ati e nt i s u n d er g oi n g a C R T d e vi c e i m pl a nt, a pr e -p e ct or al p o c k et i s m a d e r o uti n el y t o h o u s e t h e 
g e n er at or. T h e s ki n a n d s u b c ut a n e o u s pl a n e s ar e di s s e ct e d t o e x p o s e t h e f a s ci a of t h e p e ct or ali s m aj or. A 
s m all pi e c e ( 0. 5 -1. 0 g) of t h e c h e st m u s cl e ( P e ct or ali s m aj or) bi o p s y will b e t a k e n f or m et a b ol o mi c s a n al y si s, 
a n d mit o c h o n dri al p h y si ol o g y. A p pr o xi m at el y 1 0 0 m g of m u s cl e ti s s u e will b e i m m e di at el y u s e d t o e v al u at e 
mit o c h o n dri al
 c a p a cit y
 a n d
 bi o e n er g eti c
 effi ci e n c y
 u si n g
 hi g h -r e s ol uti o n
 r e s pir o m etr y
 a n d
 
s p e ctr ofl u or o m etr y, a s w e h a v e d e s cri b e d pr e vi o u sl y [ s u p 1, 2].  T h e r e m ai n d er of t h e m u s cl e ti s s u e will b e 
i m m e di at el y bl ott e d, fr o z e n i n li q ui d nitr o g e n, a n d st or e d at -8 0 C.  M u s cl e mit o c h o n dri al c o nt e nt will b e 
e v al u at e d  b y  l a b or at or y -b a s e d  e n z y m ati c  a s s a y s,  pr ot ei n  e x pr e s si o n,  a n d  m R N A  a b u n d a n c e  [ s u p  3].   
M a s s  s p e ctr o m etr y  a n d  N M R  s p e ctr o s c o p y  will  b e  u s e d  t o  d et e ct  a n d  q u a ntit at e  m et a b olit e s  i n  m u s cl e  
ti s s u e l y s at e s [ s u p 4]. M u s cl e bi o p s y m a y c a u s e bl e e di n g. W e will u s e r e a dil y a v ail a bl e c a ut er y t o st o p t h e 
bl e e di n g.  
 
D e vi c e pr o gr a m mi n g a n d f oll o w -u p  
D e vi c e p a ci n g s etti n g s will b e pr o gr a m m e d i n t h e D D D R m o d e at 6 0 t o 1 5 0 b e at s/ mi n ut e s . T h e C R T -O N or 
C R T -O F F will b e pr o gr a m m e d a c c or di n g t o t h e r a n d o mi z ati o n.  P ati e nt s will b e c all e d at 1 -m o nt h t o e v al u at e 
f or p ot e nti al p o c k et di s c o m f ort a n d pr o c e d ur e -r el at e d c o m pli c ati o n s. P ati e nt s will r et ur n f or foll o w -u p at 6-
m o nt h  (i niti al pr o gr a m C R T -O N  or C R T -O F F ) a n d 1 2-m o nt h  ( cr o s s o v er pr o gr a m C R T - O N or C R T -O F F ). 
P eri p h er al b l o o d will b e dr a w n f or m et a b ol o mi c st u dy , a n d e c h o c ar di o gr a p h y, 6- mi n ut e w al k, K C C Q, a n d 
N Y H A cl a s s will b e a s s e s s e d at e a c h f oll o w -u p. P ati e nt s will u n d er g o st a n d ar d cli ni c al c ar e t o m a xi mi z e 
C R T r e s p o n s e.  De vi c e f u n cti o n will b e cl o s el y m o nit or e d o n a d ail y b a si s vi a a r e m ot e m o nit ori n g s y st e m. 
P ati e nt s will c o nti n u e a st a bl e m e di c al t h er a p y t hr o u g h e ntir e st u d y  t o mi ni mi z e c o nf o u n di n g d u e t o dr u g 
c h a n g e s d uri n g t h e st u d y . A n y a dj u st m e nt i n m e di c ati o n s, if cli ni c all y n e e d e d, will b e c ar ef ull y r e c or d e d  a n d 
w e will t a k e i nt o c o n si d er ati o n t h e c o nf o u n di n g dr u g eff e ct i n a n al y si s . Aft er p ati e nt s c o m pl et e t h e st u d y, 
t h e y will b e f oll o w e d a n n u all y b y p h o n e t o a s s e s s a d v er s e e v e nt s. 
 
S c h e d ul e of E v e nt s  
 
S cr e e ni n g 
( wit hi n 9 0 
d a y s of 
i m pl a nt) 
D e vi c e 
I m pl a nt 
( D a y 0 ± 
4 d a y s)  
P o st -
I m pl a nt 
( D a y 0 ± 
4 d a y s)  
1 M o nt h 
P h o n e 
C all ( ±  7 
d a y s)  
6 M o nt h 
( ±3 0  
d a y s)  
7 
m o nt h 
( ± 7 
d a y s)  
1 2 M o nt h 
( ± 3 0 
d a y s)  
2 4, 3 6, & 
4 8 M o nt h 
P h o n e 
C all s ( ± 3 0 
d a y s)  
E c h o c ar di o gr a m  
( m a y i n cl u d e 3 D 
e c h o i m a gi n g ) 
X 2  
 
 
 
X 
 
X 
 
P h y si c al e x a m  
X 
 
 
 
X 
 
X 
 
1 2 -L e a d E C G  
X 
 
X 
 
 
 
 
 

Protocol version 10 dated June 20, 2021 
Page 12 of 27 
Six-minute walk 
test 
X 
 
 
 
X 
 
X 
 
Kansas City 
Cardiomyopathy 
Questionnaire 
(KCCQ) 
X 
 
 
 
X 
 
X 
 
Chest X-ray 
 
 
X 
 
 
 
 
 
Pregnancy test (if 
applicable) 
X1 
 
 
 
 
 
 
 
Blood collection 
For biomarker 
analysis 
X 
X 
 
 
X 
 
X 
 
Device 
programming/ 
interrogation 
 
 
X 
 
X 
 
X 
 
 SAE/AE 
Assessment 
(heart failure 
symptoms, pocket 
pain, device 
function, etc.) 
 
 
X 
X 
X 
X 
X 
X 
Medication 
Reconciliation 
X 
 
X 
X 
X 
X 
X 
 
1. Females of child-bearing potential must have a negative pregnancy test within 7 days of device implant. 
2. Imaging can be done within 12 months of implant.  
 
Metabolomic analyses 
To obtain comprehensive and confident information about the metabolome and metabolic pathways, a 
combination of different techniques are necessary because each technique covers different classes, 
masses, and concentration ranges of metabolites. Comparing quantitative 1H and 31P, NMR with 
semiquantitave mass spectrometric technologies permits better assessment of metabolite levels and 
dynamics. Selected metabolomics profiling will be done using LC/MS, GC/MS and 1H NMR technologies 
available at our Metabolomics Resources Core. The Core is staffed with experienced analytical and 
statistical/bioinformatics staff. In addition, we will employ novel whole blood 18O-stable isotope labeling 
technique – 18O-assisted GC/MS and 31P NMR, to detect subtle alterations in metabolite dynamics and 
metabolic pathways.42,73  
In addition to NT BNP, subject samples will be tested for several other specific metabolomics biomarkers.  
In HF, the plasma long-chain acylcarnitines are substantially elevated due to impaired mitochondrial 
oxidative capacity.  Simply a signal of mitochondrial dysfunction, the long-chain acylcarnitines is considered 
as a prognostic biomarker in end-stage HF and as an early detectable biomarker in mild LV dysfunction.   
Amino acids are important substrates for energy production in the heart.  The branched-chain amino acids, 
specifically valine, leucine and isoleucine are elevated in HF.  The amnio acids valine, leucine and 
isoleucine will be determined as biomarkers.  Using the new technology of whole blood 18O metabolic 
labeling, we will measure 2,3-BPG[18O] and G-3-P[18O] turnover rates that are reduced, while inorganic 
phosphate (Pi[18O]) and γ-ATP[18O] are elevated in mild LV dysfunction.       
LC/MS and GC/MS. The LC-MS analyses will be performed using a 6500 Series Accurate-Mass 
Quadrupole Time-of-Flight LC-MS (Agilent Technologies) coupled with an Acquity UPLC System (Waters). 
Metabolomic profiling data are acquired under both positive and negative electrospray ionization conditions. 
Metabolite separation is achieved using 2 columns of differing polarity. The analysis is run against the 

Protocol version 10 dated June 20, 2021 
Page 13 of 27 
Figure 13. Metabolite dynamics in HF patients whole blood as assessed by 18O-
assisted 31P NMR. HF patient had lower labeling and turnover rate of 2,3-
bisphosphoglycerate (2,3-BPG[18O]), which regulates hemoglobin affinity to O2 and 
blood oxygen transport capacity. At the same time labeling, the turnover rate of 
inorganic phosphate (Pi[18O]) was increased, signifying a higher ATP hydrolysis and 
degradation rate (ATP+H218OADP+Pi[18O]). 
Figure 14. Metabolic pathways and metabolites involved in metabolic 
remodeling of myocardium in heart failure. Marked in blue are 
metabolites altered in plasma. 
METLIN metabolite database to 
give 
putative 
molecular 
identifications. 
Components 
assigned putative identification are 
further verified by comparison to 
purchased 
reference 
standards 
and MS-MS data. Acyl-carnitines 
were measured by LCMS as 
previously 
described.74 
The 
samples and calibration standards 
were analyzed on Thermo TSQ 
Quantiva 
mass 
spectrometer 
(West Palm Beach, FL). Data 
acquisition 
was 
done 
using 
selective ion monitoring (SRM). 
Data files from GC-MS analyses 
will be deconvoluted using AMDIS software; SpectConnect will be used to list and track metabolite 
peaks.42,75 The Agilent Fiehn RTL Library will be used for metabolite identifications. 
Human whole blood 18O metabolic labeling. This novel technology was developed by Dr. Petras Dzeja at 
Mayo Clinic. Blood plasma and cells carry a wealth of metabolic information about health status and 
disease biomarkers.25,76,77 Labeling of fresh whole blood will be performed by supplementing with 30% 
H2[18O] in 0.9% NaCl and labeling for 5-10 min, extracting metabolites and analyzing by 18O-assisted 
GC/MS and 31P NMR.42 Preliminary studies 
indicate that abnormal G-3-P[18O] turnover 
indicates defects in α-glycerophosphate shuttle 
and substrate supply to mitochondria.72 Figure 
13 shows an example of dysregulation of 2,3-
BPG[18O] and Pi[18O] turnover rates, which 
normally regulates nucleotide metabolism and 
hemoglobin affinity to O2 and blood oxygen 
transport capacity in a severe HF patient.  
18O-assisted 
31P NMR and 
1H NMR. This 
technology is unique to Mayo Clinic permiting 
detection 
dynamics 
of 
phosphorylated 
metabolites in human blood.42 High-resolution 
1H NMR spectra will be acquired on a Bruker 
Avance III 600 spectrometer (Avance) while 31P 
spectra are acquired with 500 MHz on a Bruker 
11 T spectrometer (Avance). Percentages of 
16O, 18O1, 18O2 and 18O3 phosphoryl species in 
phosphometabolites are proportional to the 
integrals of their respective peaks in the 31P 
NMR spectrum. Spectra peaks will be identified 
according to Chenomx NMR Suite 6.1 software 
as previously described.78,79 
 
Objectives of metabolomic measurements 
1) We will perform anatomical metabolomic mapping in correlation with the electrical conduction delay in the 
LV lateral wall. The blood samples from the coronary sinus that reflect the overall myocardial metabolism 
and from the LV lateral wall (the site of latest activation) that reflect the local metabolism will be collected 
and compared. A differential feature of regional metabolism will provide insight into fundamental 
metabolomic regulation in ventricular dyssynchrony. QRS duration is a clinical predictor of CRT response. 
We will correlate the metabolic predictive value of CRT outcomes with QRS duration.   

Protocol version 10 dated June 20, 2021 
Page 14 of 27 
2) The metabolomic mapping will characterize regional metabolism in ICM vs NICM.  
3) We will gain insight in early perturbation of myocardial metabolomic profile in patients with mildly reduced 
LVEF. Specific focus will be paid to the following metabolic pathways or markers. 
a. Myocardium primarily meets its requirements for energy through the oxidation of long-chain fatty acids 
(LCFA), where carnitine plays a key role as a carrier. The “carnitine shuttle” facilitates transfer of LCFA to 
cross mitochondrial membrane for β-oxidation.80 In HF, the plasma long-chain acylcarnitines (LCAC) are 
substantially elevated due to impaired mitochondrial oxidative capacity (Figure 7 and 14, green box).55 The 
LCAC has adverse effects on HF including an increase in cytoloic calcium loading, myocardial inflammation, 
and bioenergetics deficiency.50 Simply a signal of mitochondrial dysfunction, LCAC is considered as a 
prognostic biomarker in end-stage HF.51 With a cross-over study design, the role of LCAC will be examined 
as an early detectable biomarker in relation to LV dysfuction.  
b. Amino acids are important substrates for energy production in the heart. Through a series of reactions, 
branched-chain amino acids (BCAAs) can be converted to metabolic intermediates and fed into the 
tricarboxylic acid (TCA) cycle as potential sources of anaplerotic input (Figure 14, red box). Due to the down 
regulation of gene expression related to the BCAA degradation and utilization, specifically, valine, leucine 
and isoleucine are accumulated in HF.53,81,82 Our preliminary data (shown in Figure 6 and 10), showed that 
the level of these BCAAs were disturbed even in mild HF and was significantly lower in CRT non-
responders than in CRT responders with severe HF. Further, amino acids are not only cell building blocks, 
energy source and signaling molecules but are also regulators of gene expression and protein synthesis. 
Taken together, the BCAAs are critical metabolite markers in HF. 
c. Using the new technology of whole blood 18O metabolic labeling, we will test the hypothesis that the 2,3-
BPG[18O] and G-3-P[18O] turnover rates are reduced, while inorganic phosphate (Pi[18O]) and γ-ATP[18O] 
are elevated in mild LV dysfunction in which the impaired O2 and substrate transport and ATP synthesis 
may precede the HF symptoms (Figure 13).  
d. We will develop strong metabolic biomarkers as proposed in above to determine the prognostication with 
clinical outcomes in patients with mild HF. 
Primary clinical efficacy objective 
The  study  primary  endpoint  is  a  reduction  in  the  LV  end-systolic  volume  index (LVESVI,  reverse LV  
remodeling) in the CRT-ON group as compared to CRT-OFF group.  
Secondary clinical efficacy objectives (composite events) 
1) Hospital admission for HF; 2) Increase in NT pro-BNP level by 30% and its predictive value of 
response to CRT; 3) Sustained ventricular tachycardia or fibrillation greater than 30 seconds; 4) 
Decrease in LVEF to ≤35%; and 5) death. 
 
The analytical imprecision of natriuretic peptides depends on the assay but for NTprpoBNP it is roughly 
1.6%.  Inter and intradividual variation is roughly 30% (American Journal of Cardiology. 92(5):628-31, 
2003).  Thus values above 30% have been suggested to define significant changes in natriuretic peptides 
(Journal of Investigative Medicine. 61(6):950-5, 2013 Aug,).  This value was  shown to have prognostic 
significance in the VALHEFT trial (Journal of the American College of Cardiology. 52(12):997-1003, 2008 
Sep 16) and thus will provide an end point in our proposed analysis. 
 
Primary safety objectives 
Freedom from system-related complications in randomized participants will be calculated at 1, 6, and 12 
months post-implant to determine the safety profile, including the device pocket hematoma, pneumothorax, 
myocardial perforation, lead dislodgment, lead revision, and device-related infection. This safety profile will 
be balanced against efficacy to quantify the risk-benefit ratio. This analysis will occur when all subjects have 
completed study procedures. 
Sample Size Considerations and Statistical analysis  
The timing of primary endpoint analysis will occur when all subjects have completed study procedures. The 
primary endpoint for Aim 1 is the change in LVESVI. We propose a paired t-test, which combines both 

Protocol version 10 dated June 20, 2021 
Page 15 of 27 
randomized groups together (n=50 + 50 = 100), and compares the CRT-ON condition to the CRT-OFF 
condition. The power can be estimated based on REVERSE (LVEF ≤40%) showing a mean change of -18.4 
+/- 29.5 ml/m2. Let us assume that in the current mildly impaired EF group, the reduction will be, not -18, 
but -12. Assuming the SD is 26, the power for the one-sample paired t-test is over 99%. If we assume an 
80% attrition (n=40 + 40), the detectable difference with 80% power is 8.3 ml/m2, less than half of that 
observed in REVERSE. A secondary analysis will be a generalized linear model in which baseline, period 1, 
and period 2 values of LVESVI will be analyzed together, with the possibility of an order effect. If no order 
effect is detected, then the analysis will amount to the paired t-test already described. Data may be missing 
due to the occurrence of interim events. This would mean that the data are not missing at random. We 
propose to accommodate this by a rank-based procedure, in which the ON-OFF differences are ranked in 
both groups together. As a secondary endpoint, we will analyze the composite clinical endpoint described 
above. This endpoint will be analyzed as the time to the first of any of these component events. The 
crossover design can be accommodated by fitting the treatment variable ON-OFF as a time dependent 
binary variable that is 1 (ON) or 0 (OFF). Order effects can be accommodated by including interactions with 
time-period in the model.  
Metabolomics data analysis. The timing of metabolomics data analysis will occur when all subjects have 
completed study procedures. Data sets will be mean-centered and pareto-scaled to adjust the importance of 
high- and low-abundance metabolites to an equal level before carrying out statistical analyses.42,52,77-79,84-86 
The SIMCA-P+ 12.0 software package (Umetrics, Umea, Sweden) will be used for pattern recognition 
multivariate analysis, including unsupervised principal component analysis (PCA), supervised partial least 
squares discriminant analysis (PLS-DA), and orthogonal partial least-squares discriminant analysis (OPLS-
DA). The data will be examined with PCA scatter plot of the first two score vectors (t1-t2) in order to reveal 
the homogeneity of the data, any groupings, outliers, and trends. Then PLS-DA is applied to increase the 
class separation, simplify interpretation, and find potential biomarkers. The OPLS-DA will be used to identify 
the significantly different metabolites between groups. For validation, R2 (the fraction of variance explained 
by a component) and Q2 (the fraction of the total variation predicted by a component) values will be 
considered as measures of goodness of model and the model robustness, respectively. The value of Q2 
ranges from 0 to 1 and typically a Q2 value of greater than 0.4 is considered a good model. Most important 
metabolites will be selected according to the variable importance in the projection (VIP) values and the 
variables with VIP>1 will be considered for significantly changed metabolites. Metabolic pathway analysis 
will be performed using MetaboAnalyst 3.0 software.83 In addition, we will explore the new multivariate 
metabolomics data (80 variables on 80 subjects), and construct principal components agnostically using 
SAS PROC FACTOR. Preliminary data suggest that 5 to 10 factors will represent 60% or more of the joint 
variation among the 80 metabolites. The 5-10 factors will then be calculated on each subject, and used to 
predict group differences (using a two-sample t-test), and response to CRT therapy. In particular, we will 
then relate these principal components (PC’s) to response to CRT using multiple linear regression (for the 
LVESVI endpoint) and Cox Regression (for the Clinical endpoint).  
 
 
Safety and Adverse Events 
 
All adverse events occurring related to the procedure or protocol required testing during the study, including 
those not meeting the criteria of an Unanticipated Adverse Device Effect (UADE) will be recorded on the 
appropriate case report form.  Records of these events will be maintained and reports submitted to the FDA 
and IRB according to the regulatory requirements.  Expected clinical adverse events and nonsignificant (not 
serious) clinical adverse events will not be reported. Expected clinical adverse events and anticipated 
adverse device effects are those listed below. 
 
The safety and clinical performance of market-released CRT systems have been demonstrated through 
previous clinical studies. All products used within this study are FDA approved and market-released. 
Therefore, it is not anticipated that subjects enrolled in this study will be exposed to any risks beyond those 
normally associated with CRT systems, transvenous lead systems, or their implant procedure. Because 
only patients who have existing ICD systems or who meet clinical indications for ICD implant will receive 

Protocol version 10 dated June 20, 2021 
Page 16 of 27 
CRT-D implant, there is no additional risk for these patients aside from the CRT risks described here for all 
patients in this study. With the exception of the risks associated with the device and implant procedure 
implant, the risks associated with metabolomics blood draw are considered minimal, Therefore, the risk 
analysis in this section is specific to the implantation, management and therapy from the implanted CRT 
system. The subject will remain in the study for intention to treat analysis, but the investigator will avoid any 
procedures that may be determined harmful.Study subjects will receive a CRT device with the associated 
three leads (one right atrial, one right ventricular and one left ventricular lead).  
 
The risks normally associated with the implant of the CRT system include, but are not limited to the 
followings: pain, swelling and/or bruising around the device, collection of blood (hematoma) or fluid 
(seroma) in the tissues, infection, allergy to the device material, movement of the device from its original 
location, or generator erosion through the skin over time. The risks normally related to the use of 
transvenous lead systems include, but are not limited to the following: cardiac perforation, cardiac 
tamponade, 
pneumothorax, 
pericardial 
effusion, 
pericarditis, 
valve 
damage, 
thromboembolism, 
endocarditis, ventricular fibrillation or other arrhythmias, non-cardiac muscle and/or nerve stimulation, 
myopotential sensing, hemothorax, thrombosis, heart failure, tissue necrosis or infection. The pacemaker, 
software, or programmer may malfunction and fail to detect and/or treat arrhythmia leading to death. Other 
potential adverse events related to the leads include, but are not limited to, the following: lead insulation 
failure, lead breakage difficulty removing the lead, poor connection to the device, elevated thresholds, lead 
damage lead disloged from its original location that may require that the lead be replaced or repositioned. 
Additional risks associated with the CRT system include, but are not limited to:  
• 
The placement of the LV lead requires the use of a dye to conduct the venogram. 
With the venogram there is a risk of an allergic reaction to this x-ray dye that  
Could result in serious injury (such as shock or death). There is also a chance that the dye may 
damage the kidneys. 
• 
Exposure to an increased amount of radiation through fluoroscopy and x-rays. 
• 
Vein wall rupture, dissection and perforation of the coronary sinus, cardiac vein, 
• 
Myocardium or pericardium from the left ventricular pacing lead or implant tool. 
• 
Due to the nature of the coronary sinus anatomy, a chronically implanted coronary sinus lead may 
be more difficult to remove than a traditional endocardial lead. 
• 
There may be risks associated with worsening of the subject’s heart failure. 
• 
Complications during surgery include, but are not limited to: stroke, heart attack, blood clots, 
bleeding, and death. 
• 
The Cardiac Resynchronization Therapy (CRT) is considered investigational and/or outside current 
medical guidelines in qualifying patients and may be no more effective or less effective than optimal 
medical therapy. 
• 
Study-related blood tests (e.g. BNP) might cause mild discomfort or irritation or some discomfort 
following blood draw. 
• 
Contrast agent allergy is usually infrequent and symptoms are shortness of breath, hives, itching, 
back pain, chest pain, or rash; allergic and potentially life threatening hypersensitivity reactions may 
occur rarely, including anaphylactoid and/or anaphylactic reactions, shock, bronchospasm, tongue 
and/or throat swelling, decreased oxygen saturation, and loss of consciousness. 
 
Risk Minimization 
The potential risks associated with the commercially available CRT implant were identified and have been 
successfully mitigated from clinical practice. Any potential risks associated with this study are further 
minimized by selecting qualified investigators. A Data Monitoring Committee will be formed to review safety 
issues as part of the study. In addition, investigators will be actively involved in the implantation and follow-
up of the subjects implanted with the CRT systems. Risks will be minimized by careful assessment of each 
subject prior to, during, and after implant of the CRT. Prior to implant, it is recommended subjects undergo a 
complete cardiac evaluation. After implantation, subjects will be followed at regular intervals to monitor the 
condition of the implanted system and the battery. At each protocol required follow-up, , the investigator 
must interrogate the CRT to verify appropriate CRT function and to evaluate pacing and sensing 
characteristics and to assess any adverse events. 

Protocol version 10 dated June 20, 2021 
Page 17 of 27 
 
Recording and reporting of adverse event  
 
Unanticipated Adverse Device Effect (UADE): A UADE is any serious adverse effect on health or safety 
or any life-threatening problem or death caused by, or associated with, a device if that effect, problem or 
death was not previously identified in nature, severity, or degree of incidence in the investigational plan or 
IDE application (including a supplementary plan or application), or any other unanticipated serious problem 
associated with a device that relates to the rights, safety, or welfare of subjects. 
 
Adverse Effect (Event): Any untoward medical occurrence in a subject involved in clinical study of an 
investigational device; regardless of the causal relationship of the problem with the device or, if applicable, 
other study related treatment(s). 
 
Associated with the investigational device: There is a reasonable possibility that the adverse effect may 
have been caused by the investigational device. 
 
Life-threatening adverse effect: Any adverse effect that places the subject, in the view of either the 
investigator or the sponsor, at immediate risk of death from the effect as it occurred.  It does not include a 
reaction that, had it occurred in a more severe form, might have caused death. 
 
Serious adverse effect: An adverse effect is considered “serious” if, in the view of either the investigator or 
the sponsor, it results in any of the following outcomes:  
• 
death  
• 
a life-threatening AE 
• 
inpatient hospitalization or prolongation of existing hospitalization 
• 
a persistent or significant disability/incapacity 
• 
a congenital anomaly/birth defect. 
 
Unanticipated adverse effect: Any adverse effect, the nature, specificity, severity, or frequency of which is 
not consistent with the risk information in the clinical study protocol or elsewhere in the current IDE 
application. 
General Physical Examination Findings: At screening, any clinically significant abnormality should be 
recorded as a preexisting condition.  At the end of the study, any new clinically significant 
findings/abnormalities that meet the definition of an adverse event must also be recorded and documented 
as an adverse event. 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be documented and 
reported as an unanticipated adverse device effect unless specifically instructed otherwise in this protocol.  
Any condition responsible for surgery should be documented as an adverse event if the condition meets the 
criteria for an adverse event. 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an adverse 
event in the following circumstances: 
• 
Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures for a 
preexisting condition.  Surgery should not be reported as an outcome of an adverse event if the 
purpose of the surgery was elective or diagnostic and the outcome was uneventful 
• 
Hospitalization or prolonged hospitalization required to allow efficacy measurement for the study 
• 
Hospitalization or prolonged hospitalization for therapy of the target disease of the study, unless it is 
a worsening or increase in frequency of hospital admissions as judged by the clinical investigator. 
 

Protocol version 10 dated June 20, 2021 
Page 18 of 27 
Post-study Adverse Event 
All unresolved adverse events should be followed by the investigator until the events are resolved, the 
subject is lost to follow-up, or the adverse event is otherwise explained.  At the last scheduled visit, the local 
investigator should instruct each subject to report, to the local investigator, any subsequent event(s) that the 
subject, or the subject’s personal physician, believes might reasonably be related to participation in this 
study.  The local investigator should notify the study regulatory sponsor of any death or adverse event 
occurring at any time after a subject has discontinued or terminated study participation that may reasonably 
be related to this study.  The sponsor should also be notified if the local investigator should become aware 
of the development of problems, cancer or of a congenital anomaly in a subsequently conceived offspring of 
a subject that has participated in this study. 
 
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A preexisting condition should be 
recorded as an adverse event if the frequency, intensity, or the character of the condition worsens during 
the study period. 
 
 
 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets all of the following three criteria: 
• 
Serious: Serious problems or events that results in significant harm, (which may be physical, 
psychological, financial, social, economic, or legal) or increased risk for the subject or others 
(including individuals who are not research subjects). These include: (1) death; (2) life threatening 
adverse experience; (3) hospitalization - inpatient, new, or prolonged; (4) disability/incapacity - 
persistent or significant; (5) birth defect/anomaly; (6) breach of confidentiality and (7) other 
problems, events, or new information (i.e. publications, DSMB reports, interim findings, product 
labeling change) that in the opinion of the local investigator may adversely affect the rights, safety, 
or welfare of the subjects or others, or substantially compromise the research data, AND 
• 
Unanticipated: (i.e. unexpected) problems or events are those that are not already described as 
potential risks in the protocol, consent document, not listed in the Investigator’s Brochure, or not part 
of an underlying disease. A problem or event is "unanticipated" when it was unforeseeable at the 
time of its occurrence. A problem or event is "unanticipated" when it occurs at an increased 
frequency or at an increased severity than expected, AND 
• 
Related: A problem or event is "related" if it is possibly related to the research procedures. 
 
At each contact with the subject, the investigator must seek information on adverse events by specific 
questioning and, as appropriate, by examination.  Study subjects will be routinely questioned about adverse 
effects at study visits. Information on all adverse events should be recorded immediately in the source 
document, and also in the appropriate adverse event section of the case report form (CRF) or in a separate 
adverse event worksheet.  All clearly related signs, symptoms, and abnormal diagnostic, laboratory or 
procedure results should recorded in the source document. 
 
All adverse events related to the procedure or testing required by the protocol occurring during the study 
period must be recorded.  All observed or volunteered adverse effects (serious or non-serious) and 
abnormal test findings, regardless of the treatment group if applicable or suspected causal relationship to 
the investigational device or if applicable other study treatment or diagnostic product(s) will be recorded in 
the subjects’ case history.  For all adverse effects sufficient information will be pursued and or obtained as 
to permit; an adequate determination of the outcome, an assessment of the casual relationship between the 
adverse effect and the investigational device or, if applicable other study treatment or diagnostic product.  
The clinical course of each event should be followed until resolution, stabilization, or until it has been 
ultimately determined that the study treatment or participation is not the probable cause.  Serious adverse 
events that are still ongoing at the end of the study period must be followed up, to determine the final 

Protocol version 10 dated June 20, 2021 
Page 19 of 27 
outcome.  Any serious adverse event that occurs after the study period and is considered to be at least 
possibly related to the study treatment or study participation should be recorded and reported immediately. 
 
Reporting of Unanticipated Adverse Device Effects and Unanticipated Problems 
When an adverse event has been identified, the study team will take appropriated action necessary to 
protect the study participant and then complete the Study Adverse Event Worksheet and log.  The sponsor-
investigator will evaluate the event and determine the necessary follow-up and reporting required. The 
sponsor-investigator will promptly review documented Unanticipated Adverse Device Effects and as 
necessary shall report the results of such evaluation to FDA within 10 working days and Mayo IRB within 5 
working days of initial notice of the effect.  Thereafter the sponsor-investigator will submit such additional 
reports concerning the effect as requested. A copy of this completed form will be provided to the DSMB and 
all participating sub-investigators.The completed FDA Form 3500A will be submitted to the FDA as soon as 
possible and, in no event, later than 10 working days after the sponsor-investigator first receives notice of 
the adverse effect. 
 
Medical Monitoring 
It is the responsibility of the sponsor-investigator to oversee the safety of the study.  This safety monitoring 
will include careful assessment and appropriate reporting of adverse events as noted above, as well as the 
construction and implementation of a site data and safety-monitoring plan (see Section 10 Auditing, 
Monitoring and Inspecting).  Medical monitoring will include a regular assessment of the number and type of 
serious adverse events. 
Data and Safety Monitoring Board 
A Data Safety Monitoring Board (DSMB) has been established for this study. 
 
Data Handling and Record Keeping 
Confidentiality 
Information about study subjects will be kept confidential and managed according to the requirements of the 
Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those regulations require a signed 
subject authorization informing the subject of the following: 
• 
What protected health information (PHI) will be collected from subjects in this study 
• 
Who will have access to that information and why 
• 
Who will use or disclose that information 
• 
The rights of a research subject to revoke their authorization for use of their PHI. 
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, retains 
the ability to use all information collected prior to the revocation of subject authorization.  For subjects that 
have revoked authorization to collect or use PHI, attempts should be made to obtain permission to collect at 
least vital status (long term survival status that the subject is alive) at the end of their scheduled study 
period. 
Source Documents 
Source data comprise all information, original records of clinical findings, observations, or other activities in 
a clinical trial necessary for the reconstruction and evaluation of the trial.  Source data are contained in 
source documents.  Examples of these original documents, and data records include: hospital records, 
clinical and office charts, laboratory notes, memoranda, subjects’ diaries or evaluation checklists, pharmacy 
dispensing records, recorded data from automated instruments, copies or transcriptions certified after 
verification as being accurate and complete, microfiches, photographic negatives, microfilm or magnetic 
media, x-rays, subject files, and records kept at the pharmacy, at the laboratories, and at medico-technical 

Protocol version 10 dated June 20, 2021 
Page 20 of 27 
departments involved in the clinical trial. When applicable, information recorded on the CRF shall match the 
Source Data recorded on the Source Documents. 
Case Report Forms 
Case Report Forms (CRFs) will be completed for each subject enrolled into the clinical study.   
Records Retention 
The sponsor-investigator will maintain records and essential documents related to the conduct of the study.  
These will include subject case histories and regulatory documents. 
 
The sponsor-investigator will retain the specified records and reports for: 
1. Up to 2 years after the marketing application is approved for the drug; or, if a marketing application 
is not submitted or approved for the drug, until 2 years after shipment and delivery of the drug for 
investigational use is discontinued and the FDA has been so notified.  
 
OR 
2. As outlined in the Mayo Clinic Research Policy Manual –“Retention of and Access to Research Data 
Policy” http://mayocontent.mayo.edu/research-policy/MSS_669717, whichever is longer. 
Study Monitoring, Auditing, and Inspecting 
Study Monitoring Plan 
The investigator will allocate adequate time for such monitoring activities.  The Investigator will also ensure 
that the monitor or other compliance or quality assurance reviewer is given access to all the study-related 
documents and study related facilities (e.g. pharmacy, diagnostic laboratory, etc.), and has adequate space 
to conduct the monitoring visit. 
Auditing and Inspecting 
The sponsor-investigator will permit study-related monitoring, audits, and inspections by the IRB, the 
monitor, and government regulatory agencies, of all study related documents (e.g., source documents, 
regulatory documents, data collection instruments, study data etc.).  The sponsor-investigator will ensure 
the capability for inspections of applicable study-related facilities (e.g., pharmacy, diagnostic laboratory, 
etc.). 
Participation as a sponsor-investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable compliance offices. 
Ethical Considerations 
This study is to be conducted according to United States government regulations and Institutional research 
policies and procedures. 
This protocol and any amendments will be submitted to a properly constituted local Institutional Review 
Board (IRB), in agreement with local legal prescriptions, for formal approval of the study.  The decision of 
the IRB concerning the conduct of the study will be made in writing to the sponsor-investigator before 
commencement of this study. 
All subjects for this study will be provided a consent form describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in this study.  This consent 
form will be submitted with the protocol for review and approval by the IRB for the study.  The formal 
consent of a subject, using the Approved IRB consent form, must be obtained before that subject 
undergoes any study procedure.  The consent form must be signed and dated by the subject and the 
individual obtaining the informed consent. 

Protocol version 10 dated June 20, 2021 
Page 21 of 27 
 
Potential Benefits 
The potential benefits of having the CRT include reduction in heart failure symptoms, reduced rate of 
sudden cardiac arrest, mortality and improved ventricular function.The information gained from this study 
could result in the improved management of other patients with congestive heart failure. Additionally, 
information collected from this study may assist in the design of new therapies. The arrhythmia monitoring 
features of CRT, if programmed on as determined by the investigator, may offer benefit in the event that the 
patient experiences any arrhythmia. Electronic capture of the specific arrhythmia may aid in the diagnosis 
and treatment selection for these arrhythmias. Subjects will have devices with approximately 8 years of 
remaining battery life to continue heart failure therapy.  
 
Risk-to-Benefit Analysis 
The cohort of patients defined for inclusion in this study is a result of their heart failure. The CRT offers the 
opportunity to reduce these symptoms, improve heart function, and potentially extend quality of life. These 
benefits have been proven in patients with severe heart failure. CRT has a two decade-long track record of 
published safety and efficacy for heart failure. 

Protocol version 10 dated June 20, 2021 
Page 22 of 27 
  
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