Diabetes and Lipid Accumulationand Heart Transplant
Condition: Diabetes Mellitus; Heart Failure
MeSH terms: Heart Failure
Study type: Observational

Summary: 
      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
      artery disease. IDC is the main cause of end-stage heart failure (HF) and is responsible for
      half of all heart transplants (HTx). Endocrine disorders, including diabetes, are known to be
      associated with IDC. 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. Therefore, DM can exacerbate the need for HTx, in addition, diabetic patients are
      less suitable for HTx and DM remains an independent risk factor for death even after HTx.
      Recent studies have revealed the presence of diabetic cardiomyopathy, a condition of
      myocardial dysfunction without coronary artery disease. This term was introduced for the
      first time by Rubler et al. in 1972 which highlighted patients with diabetes and congestive
      heart failure with normal coronary arteries. The pathophysiological mechanisms through which
      diabetes affects the development and progression of diabetic heart disease are not known.
      Therefore, the purpose of our study will be to evaluate, in the explanted diabetic heart, the
      presence of possible cellular alterations attributable to the diabetic disease. Furthermore,
      the progression of these lesions in the transplanted heart in diabetic patients will be
      evaluated.
    

Detailed description: 
      BACKGROUND Heart failure (HF) is an increasing disease, with a prevalence and incidence of
      about 1% and 0.15% of the general population respectively, affecting at least 300,000
      patients in the USA. However, in cases of advanced heart failure, refractory to maximal
      medical therapy, and to devices supporting the heart rhythm and cardio-circulatory function,
      the only valid therapeutic option remains the cardiac transplant. Diabetic patients with
      advanced heart failure seem to benefit from this therapy not differently as compared to
      non-diabetics. On other hand, in patients with advanced heart failure and selected for heart
      transplantation, the diabetes may condition a different degree of structural and molecular
      pathology that is not present, or present at least in a different way in non-diabetics
      selected for same HF etiology, clinical features, and HF stage. Therefore, in this study
      authors will study the anatomical-pathological, cellular, and molecular characteristics, as
      the pathways of inflammatory, oxidative, apoptotic and epigenetic expression in diabetics vs.
      non diabetics affected by idiopathic dilated cardiomyopathy (IDCM) and therefore in the
      absence of ischemic heart disease, and referred to Heart Center for cardiac transplantation.
      Authors' study hypothesis is that diabetes can condition a different degree of structural,
      cellular, and molecular pathology in patients with IDCM Vs. non-diabetic patients, due to
      excessive metabolic activity, with increased synthesis and release of inflammatory,
      oxidative, and apoptotic molecules. These investigations will be focused on microscopic,
      histological and functional analysis of the cellular metabolism of cardiac tissue extracted
      from diabetics vs. non diabetics IDCM patients, and removed during cardiac transplantation.
      However, in a subsequent ex vivo phase authors will evaluate cellular, molecular,
      inflammatory and epigenetic effectors related to the different microscopic tissue pattern
      obtained from myocardial tissue samples. From authors' investigations, these new identified
      targets of the HF molecular and cellular processes in diabetics vs. non diabetics, may be
      used in the future as specific therapeutic targets to improve clinical outcomes in IDCM
      diabetics with advanced heart failure.

      MATERIALS AND METHODS Study population Authors will enroll a population of IDCM diabetics and
      non diabetics selected to receive a heart transplant. This study will be conducted at the
      Department of Medical Sciences, at the Department of Cardiac Surgery, and at the Department
      of Biochemistry of the University of Campania "Luigi Vanvitelli". Selection, randomization
      and enrollment of patients will be carried out at the Department of Medical Sciences,
      followed by clinical follow up; Cardiac transplantation and cardiac tissue sampling will be
      performed at the Cardiosurgery Department; Molecular and cellular studies will be conducted
      at the biochemistry department. The follow-up will be 12 months. The diabetic pathology will
      be diagnosed according to the international guidelines of the American Heart Association.

      Inclusion criteria: patients aged > 18, <75 years, with indication to receive a heart
      transplant (survival score for accepted heart failure accepted (HFSS) at high risk, peak VO2
      <10 ml / kg / min after reaching the anaerobic threshold; arrhythmias recurrent symptomatic
      ventricles refractory to medical treatment, ICD and surgical), affected by IDCM with heart
      failure in NYHA class III / IV refractory to maximal medical therapy; diabetic and
      non-diabetic patients Exclusion criteria: contraindication to receiving cardiac
      transplantation; non-idiopathic dilated cardiopathy (valvulopathies, ischemic-infarct
      cardiopathy, etc.), acute myocardial infarction, acute heart failure, neoplastic disease, and
      chronic diseases that may influence the inflammatory profile both systemic and cardiac
      (cancer, chronic intestinal inflammation, hepatitis, AIDS) , and a life expectancy <6 months.
      All patients will be included in the study after signing informed consent to participate in
      the study. Routine analysis will be performed upon enrollment in the study, before cardiac
      transplantation and follow-up. During the follow-up (figure 1) clinical examinations, routine
      ecg and echocardiography will be performed regularly. Molecular study and cell study will be
      performed on myocardial tissue from explanted hearts. The study will be performed according
      to the Helsinki declaration.

      Intervention In this observational study, authors will evaluate a cohort of consecutive
      patients (diabetic vs. non-diabetic) affected by IDCM and heart failure in class III / IV
      NYHA refractory to maximal medical therapy and treated at the Division of Cardiac Surgery of
      the University of Campania "Luigi Vanvitelli "by cardiac transplantation. The study will be
      conducted in three different parts: human study, ex vivo cell study, molecular study.

      Human study: conducted in the Department of Medical Sciences and Cardiac Surgery, the
      enrolled patient will be treated by heart transplant, according to the international
      guidelines governing cardiac transplantation. After cardiac transplantation, a biopsy of
      myocardial tissue of the removed heart will be performed. The intervention will be conducted
      at the Cardiosurgery Division of the "Luigi Vanvitelli" University of Campania.

      Cardiac tissue analysis A portion of muscle tissue (50 grams) will be removed from the
      explanted heart, from which 3 portions will be obtained: a portion will be incorporated in
      the OCT compound and frozen in liquid nitrogen for immunohistochemical analysis, a second
      portion will be immediately frozen in nitrogen liquid and stored at -70 ° C for the isolation
      of RNA, and a third portion will be weighed, cut into small pieces (2 mm3) and transferred to
      a 12-well plate. Based on the weight of the tissue, serum-free DMEM (2 ml / g) will be added
      to the well and incubated at 37 ° C in a mild-fluctuated CO2 incubator. At 3 hours, the
      conditioned soils will be collected and centrifuged at 4 ° C for 10 minutes. The supernatants
      from cultures of epiphonic and subcutaneous adipose tissue will be stored in aliquots at -70
      ° C for the measurement of inflammation mediators released by ELISA.

      Blood samples

      Blood collection will be carried out on the morning of surgery and during the follow-up
      phases by peripheral venous blood taken in tubes without pyrogen with or without EDTA as
      anticoagulant. For plasma, the EDTA tubes will be placed on melted ice, then centrifuged
      within 20 minutes at 1500 g for 10 minutes at 4 ° C. The plasma will be stored in aliquots at
      80 ° C for all ELISA tests. Serum glucose, lipid panels and inflammatory markers will be
      analyzed in the University of Campania's Biochemistry Laboratory.

      Inflammatory markers

      The authors will analyze the mediators of plasma and cardiac inflammation with ELISA (R & D
      systems) according to the procedure recommended by the manufacturer. ELISA standard kit they
      will be used for IL-6 measurements, and highly sensitive ELISA kits for TNF-alpha and IL-1
      measurements. Intra-assay variability will be set at 10%, while inter-assay variability will
      be 15%.

      RNA analysis and Real-Time Reverse Transcription

      Samples of myocardial tissue will be minced in a TriZol reagent (Invitrogen) and homogenized
      completely on ice. The total RNA will be extracted from the chloroform and purified twice
      through the mini RNAasy columns. After the DNase treatment on a column, the RNA will be
      eluted with RNase-free water. Transcripts encoding various inflammatory mediators will be
      measured by the TaqMan real-time reverse-polymerase-RT (PCR) chain reaction with the TaqMan
      Gold RT-PCR and the PRISM 7700 Sequence Detection System (Applied Biosystems). PCR primers
      and TaqMan probes will be obtained from Applied Biosystems and optimized according to the
      manufacturer's protocol. The PCR reaction conditions will be at 48 ° C for 30 minutes, at 95
      ° C for 10 minutes, followed by 40 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute.
      The GAPDH transcripts will be amplified in a separate tube to normalize the variance in the
      input RNA. The mRNA in various samples will be estimated by the relative standard method with
      a series of dilutions of RNA from human vascular cells or from leukocytes.

      Immunohistochemistry The authors will obtain frozen sections (10 m), which will be air-dried
      for 15 minutes and immersed in xylene for 10 minutes to remove the fat. The sections will
      then be hydrated in decreasing degrees of alcohol and stained with hematoxylin and eosin. The
      selected serial sections will be immunosimochemical with the Universal Elite ABC (Vector
      Laboratories) kit according to the manufacturer's protocol. Briefly, the sections will be
      incubated with 0.3% H2O2 in methanol for 30 minutes, followed by a block with horse serum or
      5% goat. After washing in PBS, the sections will be incubated with primary antibodies for 1
      hour in a wet chamber. Subsequently, the slides will be incubated with secondary antibodies
      for 30 minutes followed by avidin-biotin for 30 minutes. The sections will then be exposed to
      DAB and counterstained with hematoxylin. The following antibodies will be used: CD3
      (Tlymphocyte, 1:50, Novocastra), CD68 (monocytes / macrophages, 1: 100, Dako) and triptases
      (mast cells, 1:50, Novocastra).

      Follow-up After being discharged from the hospital, all patients will be required to carry
      out control visits, as indicated by the authors on the management of patients post-transplant
      cardiac, at the Division of Cardiac Surgery of the University of Campania "Luigi Vanvitelli
      "and the sixth division of Internal Medicine of the University of Campania" Luigi Vanvitelli
      ". All patients will be monitored for 12 months after follow-up, by clinical evaluation (ECG,
      stress test, echocardiogram) to maintain HbA1c levels <7%, fasting glycemia between 90 and
      140 mg / dl and post-prandial glycemia <180 mg / dl, as indicated in the guidelines for the
      management of diabetic and post-CABG patients. In the 12 months of follow-up, patient
      management will be conducted by telephone interview, physical examination (at discharge and
      3, 6 and 12 after cardiac transplantation), ecg and echocardiography (at discharge and 3, 6
      and 12 after cardiac transplantation); CMRI will be conducted at baseline and 12 months after
      CABG. Similarly, the bio-humoral evaluation will be conducted during all the follow-up
      phases.

      Statistical Analysis The 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 between diabetics vs. non-diabetics. Cox regression will be used to construct the
      predictive model of mortality. The risk ratio for mortality will be adjusted for age, BMI,
      cholesterol, LDL, triglycerides and aspirin, ticlopidine, anti-aggregating agents,
      beta-blockers, ACE inhibitors or sartans, antidiabetic drugs, statins, etc. present at the
      time of hospitalization for cardiac transplantation. Analysis of survival after cardiac
      transplantation will be performed using the Kaplan-Meier curve and Cox regression method.
      Mortality curves will be obtained separately for diabetic patients compared to non-diabetic
      patients, and then compared using the log-rank test. All tests will be considered significant
      if with a value of p <0.05. All analyzes will be conducted in 2 populations: diabetic vs
      non-diabetic patients after cardiac transplantation. For all analyzes the SPSS program will
      be used (version 21, IBM SPSS).
    

Intervention name(s): patients treated by HTx
Intervention type(s): Other
Intervention description(s): From removed and frozen hearts, authors will conduct a tissue and molecular analysis of possible mechanisms T2DM linked.
Observational model: Cohort
Time perspective: Other
Sampling method: Non-Probability Sample
Study population: 
        Two main groups according to whether patients did or did not have pre-transplantation
        diabetes. Patients with DM for at least 6 months before HTX, with optimal glycemic control
        (HbA1c <7.0), BMI <30kg/m2, without severe secondary end-organ disease will be included.
        Patients with endomyocardial biopsy specimens consistent with ISHLT Grade 2R considered
        positive for rejection, increased T4/T8 ratio, positive IgM and/or IgG Cytomegalovirus
        antibodies, and with post-HTX diabetes will be excluded.
      
Biospecimen description: 
      The patients received in the past heart transplant. In the removed and frizzed hearts authors
      will extract portions of septal wall. From this tissue authors will analyze inflammatory
      markers and microRNAs involved in the progression of cardiac disease.