Failing Heart Transplants and Rejection—A Cellular Perspective

The median survival of patients with heart transplants is relatively limited, implying one of the most relevant questions in the field—how to expand the lifespan of a heart allograft? Despite optimal transplantation conditions, we do not anticipate a rise in long-term patient survival in near future. In order to develop novel strategies for patient monitoring and specific therapies, it is critical to understand the underlying pathological mechanisms at cellular and molecular levels. These events are driven by innate immune response and allorecognition driven inflammation, which controls both tissue damage and repair in a spatiotemporal context. In addition to immune cells, also structural cells of the heart participate in this process. Novel single cell methods have opened new avenues for understanding the dynamics driving the events leading to allograft failure. Here, we review current knowledge on the cellular composition of a normal heart, and cellular mechanisms of ischemia-reperfusion injury (IRI), acute rejection and cardiac allograft vasculopathy (CAV) in the transplanted hearts. We highlight gaps in current knowledge and suggest future directions, in order to improve cellular and molecular understanding of failing heart allografts.

RyR2 and Calcium Release in Heart Failure

Heart Failure (HF) is defined as the inability of the heart to efficiently pump out enough blood to maintain the body's needs, first at exercise and then also at rest. Alterations in Ca2+ handling contributes to the diminished contraction and relaxation of the failing heart. While most Ca2+ handling protein expression and/or function has been shown to be altered in many models of experimental HF, in this review, we focus in the sarcoplasmic reticulum (SR) Ca2+ release channel, the type 2 ryanodine receptor (RyR2). Various modifications of this channel inducing alterations in its function have been reported. The first was the fact that RyR2 is less responsive to activation by Ca2+ entry through the L-Type calcium channel, which is the functional result of an ultrastructural remodeling of the ventricular cardiomyocyte, with fewer and disorganized transverse (T) tubules. HF is associated with an elevated sympathetic tone and in an oxidant environment. In this line, enhanced RyR2 phosphorylation and oxidation have been shown in human and experimental HF. After several controversies, it is now generally accepted that phosphorylation of RyR2 at the Calmodulin Kinase II site (S2814) is involved in both the depressed contractile function and the enhanced arrhythmic susceptibility of the failing heart. Diminished expression of the FK506 binding protein, FKBP12.6, may also contribute. While these alterations have been mostly studied in the left ventricle of HF with reduced ejection fraction, recent studies are looking at HF with preserved ejection fraction. Moreover, alterations in the RyR2 in HF may also contribute to supraventricular defects associated with HF such as sinus node dysfunction and atrial fibrillation.

Nanoscale Organisation of Ryanodine Receptors and Junctophilin-2 in the Failing Human Heart

The disrupted organisation of the ryanodine receptors (RyR) and junctophilin (JPH) is thought to underpin the transverse tubule (t-tubule) remodelling in a failing heart. Here, we assessed the nanoscale organisation of these two key proteins in the failing human heart. Recently, an advanced feature of the t-tubule remodelling identified large flattened t-tubules called t-sheets, that were several microns wide. Previously, we reported that in the failing heart, the dilated t-tubules up to ~1 μm wide had increased collagen, and we hypothesised that the t-sheets would also be associated with collagen deposits. Direct stochastic optical reconstruction microscopy (dSTORM), confocal microscopy, and western blotting were used to evaluate the cellular distribution of excitation-contraction structures in the cardiac myocytes from patients with idiopathic dilated cardiomyopathy (IDCM) compared to myocytes from the non-failing (NF) human heart. The dSTORM imaging of RyR and JPH found no difference in the colocalisation between IDCM and NF myocytes, but there was a higher colocalisation at the t-tubule and sarcolemma compared to the corbular regions. Western blots revealed no change in the JPH expression but did identify a ~50% downregulation of RyR (p = 0.02). The dSTORM imaging revealed a trend for the smaller t-tubular RyR clusters (~24%) and reduced the t-tubular RyR cluster density (~35%) that resulted in a 50% reduction of t-tubular RyR tetramers in the IDCM myocytes (p < 0.01). Confocal microscopy identified the t-sheets in all the IDCM hearts examined and found that they are associated with the reticular collagen fibres within the lumen. However, the size and density of the RyR clusters were similar in the myocyte regions associated with t-sheets and t-tubules. T-tubule remodelling is associated with a reduced RyR expression that may contribute to the reduced excitation-contraction coupling in the failing human heart.

Abstract P386: Supplementation Of Tyrode With Fatty Acids And 3-hydroxybutyrate Improves Adult Cardiomyocytes Contractility Of Failing Human Hearts.

Due to its high energy consumption and limited ability to store ATP, the heart is highly dependent of exogenous metabolic substrates. Prior in vivo studies have reported that the development of heart failure is accompanied by a transition from the normal preferential metabolism of free fatty acids (FFA) to increases in glucose utilization and even ketone bodies, which normally provide a modest contribution to energy balance. However, the functional significance of the upregulated ketone metabolism in the failing heart is poorly understood. Recognizing that nearly all prior studies examining isolated cardiomyocyte physiology have used glucose as the sole metabolic substrate, we initiated studies to examine the impact of alternative metabolic substrates on contractility in isolated human cardiomyocytes. To understand the role of substrate alteration cardiomyocyte functionalities, we employed freshly isolated adult human left ventricular cardiomyocytes from 11 non-failing hearts (NF) obtained from organ donors and 13 failing hearts (HF) obtained from transplant recipients. Cardiomyocytes were resuspended in a conventional 5mM Glucose Tyrode solution with alternative substrates (Glucose, FFA, R-3-OHB or Mix (Glucose + FFA + 3-OHB)). Myocytes were field stimulated at 1 Hz and sarcomere length, fractional shortening, contraction velocity and relaxation velocity were measured using a video-based sarcomere length detection system (IonOptix Corp). Studies using isolated cardiac myocyte contractility as readout confirm that myocytes from NF human hearts are omnivorous: high levels of myocyte fractional shortening (FS) can be achieved under unstressed conditions (1 Hz, unloaded) with any substrate (FS Glucose : 0.1315±0.012; FS FFA : 0.1428±0.0127; FS 3OHB : 0.1343±0.014; FS MIX : 0.15467±0.02). In the failing heart, glucose alone is insufficient to produce normal unstressed myocyte fractional shortening (FS Glucose : 0.088±0.009***, p<0.001 compare to NF). However, in failing myocytes, supplementation of physiological levels of glucose with FFA or ketones each enhances myocyte contractility and rates of shortening and re-lengthening (FS FFA : 0.109±0.0127; FS 3OHB : 0.107±0.012; FS MIX : 0.112±0.016). These results suggest that future comparisons of NF vs. HF human myocyte contractility should include conditions with a physiological mix of metabolic substrates.