Cardiac Mitochondrial Function in Explanted Human Hearts

Study Overview
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Brief Summary
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Background: Treatment of heart failure has improved considerably in the past decades. Despite this improvement, the disease may progress into an end-stage ultimately leaving the physicians with no other treatment option than heart transplantation (HTx). There are multiple etiologies underlying heart failure. Cardiomyopathy is the leading cause for HTx in any age-group with coronary artery disease being the second most common cause in adult patients. Alterations in the mitochondrial function have been recognized as key factors in heart failure. During the transplant procedure the diseased heart is removed, providing a unique opportunity to collect samples eligible for thorough mitochondrial examination. Hopefully, the knowledge gained from this investigation will contribute with important insights in the diseased myocardial energy metabolism. Such knowledge may pave the way for development of treatments targeting both energy substrate supply for adenosine-triphosphate generation produced by the mitochondria as well as mitochondrial function in the failing heart. Hypothesis: The pathological myocardial function seen in heart failure is related to dysfunctional cardiac mitochondria Objective: To examine if cardiac mitochondrial function in end-stage heart failure of multiple etiologies is inferior to mitochondrial function in transplanted hearts with no signs of rejection or vasculopathy. Design: Myocardial mitochondrial function analyzed from 24 explanted hearts will be compared to endomyocardial biopsies from 20 HTx patients at scheduled biopsies (1 or 2 years after implantation).

Detailed Description
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Background: Treatment of heart failure has improved considerably in the past decades. Despite this improvement, the disease may progress into an end-stage ultimately leaving the physicians with no other treatment option than heart transplantation (HTx). There are multiple etiologies underlying heart failure. Cardiomyopathy is the leading cause for HTx in any age-group with coronary artery disease being the second most common cause in adult patients. Alterations in the mitochondrial function have been recognized as key factors in heart failure. The understanding of the complex interaction of the mitochondria in regulation of the metabolism and cellular apoptosis has brought new perspectives to research in heart failure. It is now known that the myocardial mitochondrial density changes and their function and integrity is impaired during heart failure. During the transplant procedure the diseased heart is removed, providing a unique opportunity to collect samples eligible for thorough mitochondrial examination. The collected myocardial tissue samples will be evaluated with High Resolution Respirometry, examining the glucose coupled respiratory capacity of permeabilized myocardial fibers. The respiratory capacity in the diseased fibers will be compared to the respiratory capacity in fibers from coronary healthy HTx patients transplanted 1 to 2 years prior to acquisition of the fibers. Hopefully, the knowledge gained from this investigation will contribute with important insights in the diseased myocardial energy metabolism. Such knowledge may pave the way for development of treatments targeting energy substrate supply for adenosine-triphosphate generation produced by the mitochondria as well as mitochondrial function in the failing heart. Hypothesis: The pathological myocardial function seen in heart failure is related to dysfunctional cardiac mitochondria. Objective: To examine if cardiac mitochondrial function in end-stage heart failure of multiple etiologies is inferior to mitochondrial function in transplanted hearts with no signs of rejection or vasculopathy. Design and Endpoint: Myocardial mitochondrial function analyzed from 24 explanted hearts will be compared to endomyocardial biopsies from 20 HTx patients at scheduled biopsies (1 or 2 years after implantation). Endpoints: 1) Mitochondrial respiratory capacity. 2) Mitochondrial complex function, outer membrane integrity and mitochondrial content. Methods: High-resolution respirometry: High-resolution respirometry is used to measure mitochondrial respiratory capacity in endomyocardial biopsies. After appropriate preparation, two biopsies are transferred to an oxygraph (Oxygraph-2k; Oroboros, Innsbruck, Austria) for high resolution respirometry. Mitochondrial respiratory capacity will be analyzed in a step-by-step manner using titrations of substrates and inhibitors to evaluate glucose coupled respiration in the fibers. High-resolution respirometry analysis is performed within 8 hours after the biopsy has been taken. After analysis the tissue will be snap-frozen in liquid nitrogen and stored in a research biobank at -80 degrees celsius until examination of citrate synthase activity is carried out. Hereafter, any remaining tissue will be destroyed. Electron microscopy: A sample from each biopsy used for high-resolution respirometry will be used for electron microscopy (EM) to evaluate mitochondrial volume density (MitoVD) and integrity. Muscle samples are fixated for 24 hours in glutaraldehyde and washed 4x15 minutes with Na-cacodylate buffer before being casted in Epon. The Epon casted tissue will be stored in a research biobank until EM analysis. Ultra-thin sections of the Epon-blocks (60 nm) are cut in three depths and dyed with uranyl acetate and lead citrate. Imaging is performed with an EM 208 transmission electron microscope and a Megaview III camera. All fibers are photographed at 10.000 × magnification in a randomized order. MitoVD is estimated from mitochondrial fractional area and only distinct fibers will be used in the final analysis. Statistics: Normally distributed data will be presented as mean ± standard deviation; non-normally distributed data will be presented as median and interquartile range. Categorical data are presented as absolute values or percentages. Histograms and Q-Q plots will be used to check continuous values for normality. Between-group differences will be assessed by t-test for normally distributed data and Mann-Whitney U test for non-normally distributed data. Statistical significance at a p-value of <0.05. Sample size calculation: At present there are no test-retest evaluation of mitochondrial respiratory analysis in endomyocardial biopsies from explanted human hearts. However, a recent study performed at our department on myocardial biopsies demonstrated that a total sample size of 40 human subjects in a 1:1 parallel group design was able to identify differences between the two groups (unpublished data). Data collection and processing: Source data will be recorded in the patient's electronic patient record or on specific worksheets. A centralized electronic Case Report Form (CRF) will be constructed for data capture. Data will be stored until completion of the project, after which, it will be transmitted to the Danish Data Archives. Perspectives: HTx is the golden standard treatment for patients suffering from end-stage heart failure, but its limitations cannot be ignored. Firstly, the procedure is accompanied by a substantial risk and a significant percentage of patients suffer from acute graft failure. Furthermore, HTx patients have a higher risk of severe infections and cancer, and up to 50% of HTx patients suffer from cardiac allograft vasculopathy 10 years after transplantation, all of which are highly related to mortality. Hence, postponing HTx is desirable, if health and life-quality can be kept at an acceptable level. In this context, mitochondrial function seems to be pivotal, as approaches to assess mitochondrial function in the failing heart may prove to pave the way for new follow-up algorithms and even treatment targets.

Official Title
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Cardiac Mitochondrial Function in Explanted Human Hearts

Conditions
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End-stage Heart Failure

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Informed consent from the recipient Exclusion Criteria: Myocardial biopsy from the explanted heart not feasible

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| State 2 respiration (GM) | Complex I-linked respiration, induced by Malate and Glutamate. | Within 5 minutes. | 
| State 3 respiration (GM3) | Complex I-linked respiration with adenosine diphosphate (ADP), induced by Malate, Glutamate and ADP. | Within 10 minutes. | 
| State 3 respiration (GMS3) | Complex I+II-linked respiration, induced by Malate, Glutamate, ADP and Succinate. | Within 15 minutes. | 
| State 4 respiration (4o) | Complex I+II-linked respiration not linked to adenosine triphosphate production, induced by inhibition of complex V by Oligomycin. | Within 30 minutes. | 
| Residual Oxygen Consumption (ROX) | Respiration not linked to the electron transport chain, induced by inhibition of complex I by Rotenone, complex III by Antimycin A and complex V by Oligomycin. | Within 45 minutes. | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Mitochondrial function, High Resolution Respirometry, Heart Transplantation