Lipid Accumulation in Heart Transplant From Non-diabetic Donors to Diabetic Recipients
Conditions: Diabetes Mellitus, Heart Failure
Sponsor: University of Campania Luigi Vanvitelli

1 
Unique Protocol ID: DCM-AHEAD 
Brief Title: Diabetes and Lipid Accumulationand Heart Transplant 
( DCM-AHEAD ) 
Official Title: Lipid Accumulation in Heart Transplant From Non-
diabetic Donors to Diabetic Recipients 
Project manager: Raffaele Marfella 
Department of Advanced Medical and Surgical Sciences 
Tel: +390-081-5665110 
e-mail: raffaele.marfella@unicampania.it
clinical trial number: NCT03546062 
clinical trial registry: clinicaltrialgov.com 
Naples November 19, 2019 

 
2 
 
BACKGROUND 
Idiopathic dilated cardiomyopathy (IDC) is defined by the presence of left ventricular systolic 
dysfunction in the absence of an abnormal loading condition or significant coronary heart disease 
(1). IDC is the leading cause of end-stage heart failure (HF) and is responsible for half of all heart 
transplants (HTx) (1). Endocrine disorders, including diabetes, are known to be associated with 
IDCs. Diabetes mellitus (DM), which is present in 75% of patients with idiopathic IDC, is an 
independent risk factor for the development of heart failure and death in IDC (2-4). Therefore, DM 
may exacerbate the need for HTx; moreover, diabetic patients are less suitable for HTx and DM 
remains an independent risk factor for death even after HTx (5). Recent studies have revealed the 
presence of diabetic cardiomyopathy, a condition of myocardial dysfunction without coronary 
heart disease (6, 7). This term was first introduced by Rubler et al. (8) in 1972, which highlighted 
patients with diabetes and congestive heart failure with normal coronary arteries. The 
pathophysiological mechanisms by which diabetes affects the development and progression of 
diabetic heart disease are not known. Therefore, the aim of our study will be to evaluate, in the 
diabetic heart removed, the presence of any cellular alterations related to diabetic disease. In 
addition, the progression of such lesions in the transplanted heart in diabetic patients will be 
evaluated. 
 
 
 

 
3 
 
MATERIALS AND METHODS 
Study protocol 
A prospective observational study will be conducted with a follow-up duration ranging from a 
minimum of 12 months to a maximum of 36 months (Figure).
 

 
4 
 
Patients 
A cohort of consecutive patients (diabetics vs. non-diabetics) with IDCM and NYHA class III/IV 
heart failure refractory to maximum medical therapy and treated with cardiac transplantation at 
the Division of Cardiac Surgery of the University of Campania "Luigi Vanvitelli" will be evaluated 
prospectively. At the Department of Medical Sciences will be conducted the clinical follow-up for 
cardiometabolic evaluation (eg routine, glycemic compensation). The molecular and cellular study 
will be conducted at the department of biochemistry. The follow-up will be 12 months. Patients 
will be recruited aged > 18, <75 years, with indication to receive a heart transplant (survival score 
for accepted heart failure (HFSS) at high risk, peak VO2 <10 ml / kg / min after reaching the 
anaerobic threshold; recurrent symptomatic ventricular arrhythmias refractory to medical 
treatment, ICD and surgical), affected by IDCM with heart failure in class III / IV NYHA refractory to 
maximal medical therapy; diabetic and non-diabetic patients. Patients with non idiopathic dilated 
heart disease (valvular disease, ischemic-infarct heart disease, etc.) will be excluded. All patients 
will be included in the study after signing the informed consent to participate in the study. The 
study will be performed according to the Helsinki Declaration. 
Explanted heart anlysis 
Myocardial tissues from hearts removed from diabetics vs. non-diabetics will be analyzed from a 
histological, microscopic and metabolic functional point of view. From the residual portion of the 
heart removed, after routine histological analyses conducted according to the "Heart Transplant 
Clinical Guidelines 2017". (9), a portion of muscle tissue (quantity 5-10 mm3) will be removed, 
from which 3 portions will be obtained: one portion will be incorporated into the OCT compound 
for immunohistochemical analysis, a second portion will be immediately frozen in liquid nitrogen 
and stored at -70°C for WB evaluation, and a third portion will be used for quantitative evaluations 
performed by ELISA. 

 
5 
 
 
E ndo-myocardial biopsies and Follow up  
Transplanted patients will be followed through an echocardiographic evaluation (enrolment and 3-
6-12 months follow-up), to evaluate cardiac functional parameters, metabolic parameters and 
survival outcomes (death for all causes, cardiac death, re-hospitalisation for heart failure). In all 
transplanted patients endo-myocardial biopsies (EMB) are collected according to normal clinical 
practice by the "ASHI" guideline (9): weekly for the first month; every 2 weeks for the following 
month; 1 for the following 4 weeks; 1 for the following 6 weeks; every 3 months for the following 
2 years; and finally every 6 months for the following 3 years. 2-6 (usually 4, 1 to 3 mm3) fragments 
of myocardial tissue will be collected from the apical segment of the right side of the 
interventricular septum (IVS). The collected myocardial tissue will be immediately analyzed for 
histopathological evaluation of rejection. From the remaining portion of tissue 3 portions will be 
obtained: one portion will be incorporated into the OCT compound for immunohistochemical 
analysis, a second portion will be frozen immediately in liquid nitrogen and stored at -70°C for PCR 
evaluation, and a third portion will be used for quantitative evaluations performed by ELISA.  
The conservation and morpho-functional evaluation of biopsy samples will be carried out at the 
Bio-Bank of the University of Campania "Luigi Vanvitelli". 
Biopsies analysis 
PCR. 
Total RNA will be isolated from human heart sample homogenates using the RNeasy Mini kit 
(74106, Qiagen), according to the manufacturer's protocols "Stabilization of RNA in collected 
animal tissues" and "Purification of total RNA from animal tissues". RNA concentration and purity 
will be determined using the NanoDrop 2000c (Thermo Fisher Scientific) spectrophotometer. 
Genomic DNA (gDNA) contamination will be removed from RNA samples prior to the Reverse 
Trascription (RT) phase, performed on the Gene AMP PCR System 9700 (Applied Biosystems) 

 
6 
 
using the QuantiTect Reverse Transcription Kit (205311, Qiagen), according to the "Reverse 
Transcription with Elimination of Genomic DNA for Quantitative, Real-Time PCR" protocol. The 
final step for real-time PCR analysis (qPCR) will be performed on the CFX96 Real-time System 
C1000 Touch Thermal Cycler (BIORAD). The analysis will be performed according to the "Two-
Step RT-PCR (Standard Protocol)" protocol, using the QuantiTect SYBR Green PCR Kit (204143, 
Qiagen) and specific QuantiTect Primer Assays (249900, Qiagen) for each gene tested. Relative 
quantization of gene expression will be performed using the 2^-ΔΔΔΔCt method, using GAPDH as 
the cleaning control gene. P values will be calculated on the basis of a student t-test or one-way 
variance analysis (ANOVA), followed by Dunnett's post hoc test; P < 0.05 will be considered 
significant.  
Immunofluorescence. Confocal laser scanning microscopy. 
The analysis of immunofluorescence in sections of the myocardium will be performed according to 
the manufacturing protocol using the laser scanning confocal microscope (LSM 700; Zeiss). In 
particular, the complete removal of the paraffin will be carried out before proceeding to the staining 
of the section. For this purpose, the slides will be deparaffinated by passing them through xylenes, 
alcohols and water. An antigen recovery buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) 
will be added to the dewaxed and rehydrated sections and boiled in the microwave for 20 minutes. 
After 20 minutes, immunofluorescence staining will be performed using specific antibodies against 
PPAR-alpha (1µg/ml) (polyclonal rabbit, NB600-636, Novusbio, Abingdon, UK), PPAR-gamma 
(1µg/ml) (mouse monoclonal, orb69095, Biorbyt, Cambridge, UK) or against SREBP1 (1µg/ml) 
(polyclonal rabbit, ab28481, Abcam, Cambridge, UK). Secondary antibodies will be conjugated 
with Alexa Fluor 633 or Alexa Fluor 488 (Life Technologies Italia, Monza, Italy). Nuclear contrast 
staining will be performed using the VECTASHIELD® mounting medium with DAPI (H-1200, 
Vector Laboratories, Burlingame, USA). 
Immunohistochemistry. 

 
7 
 
Oil Red O (Solvent Red 27, Sudan Red 5B, C.I. 26125, C26H24N4O) will be used for the 
assessment of the accumulation of intramyocytic lipids. 
Statistical analysis 
Study population groups (diabetics vs. non-diabetics) will be compared using the Pearson test for 
categorical variables and the Kruskal-Wallis test for continuous variables. Candidates for admission 
to the multivariate model will be identified by focusing on factors that will differ significantly (P 
value < 0.05) in the univariate analysis among diabetic vs non-diabetic patients. Cox regression will 
be used to construct the predictive model of mortality. The risk ratio for mortality will be adjusted 
by age, BMI, cholesterol, LDL, triglycerides and aspirin, ticlopidine, anti-aggregants, beta-blockers, 
ACE inhibitors or tailors, antidiabetic drugs, statins, etc. present at the time of admission to 
hospital for cardiac transplantation. The analysis of survival after cardiac transplantation will be 
performed using the Kaplan-Meier curve and the Cox regression method. Mortality curves will be 
obtained separately for diabetic and non-diabetic patients, and then compared using the log-rank 
test. All tests will be considered significant if they have a value of p<0.05. All tests will be 
conducted in 2 populations: diabetic vs non-diabetic patients after cardiac transplantation. The 
SPSS program (version 21, IBM SPSS) will be used for all analyses.  
 
 

 
8 
 
References 
 
1. 1.Hosenpud JD, Bennett LE, Keck BM, et al. The registry of the International Society for 
Heart and Lung Transplantation: Eighteenth official report  2000. J Heart Lung Transplant 
2001;20:805–15. 
 
2. L López-Sainz Á, Barge-Caballero E, Barge-Caballero G, Couto-Mallón D, Paniagua-Martin 
MJ, Seoane-Quiroga L, Iglesias-Gil C, Herrera-Noreña JM, Cuenca-Castillo JJ, Vázquez-
Rodríguez JM, Crespo-Leiro MG. Late graft failure in heart transplant recipients: incidence, 
risk factors and clinical outcomes. Eur J Heart Fail. 2018;20:385-394.  
 
3. Saraiva J, Sola E, Prieto D, Antunes MJ. Diabetes as an outcome predictor after heart 
transplantation. Interact Cardiovasc Thorac Surg. 2011;13:499-504. 
 
4. Jalowiec A, Grady KL, White-Williams C. Heart Transplant Outcomes in Patients With 
Pretransplant Diabetes Mellitus. Am J Crit Care. 2017;26:482-490. 
 
5. Morgan JA, John R, Weinberg AD, Colletti NJ, Mancini DM, Edwards NM.Heart 
transplantation in diabetic recipients: a decade review of 161 patients at Columbia 
Presbyterian. J Thorac Cardiovasc Surg. 2004;127:1486-92. 
 
6. Meagher P, Adam M, Civitarese R, Bugyei-Twum A, Connelly KA. Heart Failure With 
Preserved Ejection Fraction in Diabetes: Mechanisms and Management. Can J Cardiol. 
2018;34:632-643. 
 
7. Jia G, Hill MA, Sowers JR. Diabetic Cardiomyopathy: An Update of Mechanisms 
Contributing to This Clinical Entity. Circ Res. 2018;122:624-638. 
 
8. Rubler S, Dlugash J, Yuceoglu YZ, Kumral T, Branwood AW, Grishman A. New type of 
cardiomyopathy associated with diabetic glomerulosclerosis. Am J Cardiol. 1972;30:595-
602. 
 
9. Clinical Guidelines for Adult Heart Transplantation. The Journal of Heart and Lung 
Transplantation, Vol 35, No 1, January 2016 Revised: Date: September 2017 

 
9