Reduction of cardiac injury in DCD hearts with prolonged ischemic period: Role of MPTP opening and calpain activation

终末期心力衰竭(HF)患者需要机械循环支持，如果符合条件，还需要心脏 移植(HTX)。供体心脏的供应已经达到了一个平台期，因为它是目前唯一的供体来源。 心脏由不可逆的完全性脑损伤患者组成(脑死亡后捐赠，DBD)。然而， 符合条件的HT接受者的数量呈指数级增长。不幸的是，由于这种供需关系 不匹配，高达20%的患者在列出HTX时死亡。因此，迫切需要扩大心脏捐赠者。 游泳池。这种供体心脏的一个潜在的新来源是来自DCD(循环死亡后捐赠)的捐赠者。DCD 捐赠者将包括肝、肺和肾在内的实体器官的移植比率提高了50%。 不幸的是，DCD方案会导致持续的热缺血时间，从而损害心肌 它可用于临床移植。因此，缺血(ISC)从DCD方案来看，潜在的心肌损伤 储存引起的损伤和再灌流(REP)相关的损伤共同代表着加重损伤的额外风险 在DCD心脏中，排除了它们在临床移植中的常规使用。执行有限数量的HTX 在严格的方案下使用DCD心脏，缺血时间非常短(&lt；20分钟)。延长可接受的范围 缺血时间(35分钟)将允许大量额外的DCD心脏用于HTX。虽然很暖和 ISC在DCD心脏中是不可避免的，可通过在DCD发病时进行干预来减少Rep损伤 代表。我们建议，开发新的策略来预防代表损伤将减少对DCD的损害 心。线粒体是心肌损伤的重要靶点和介体。线粒体通透性 过渡孔(MPTP)的打开被认为是在ISC-rep过程中诱导细胞死亡的最后一步。由于MPTP 打开主要发生在REP期间，有机会通过以下方式减少MPTP打开 在代表开始时实施干预。环孢素A(CyA)是一种经典的MPTP抑制剂，它能降低 心肌缺血再灌注后的心脏损伤。我们建议在REP开始时给予CyA可以 减少DCD心脏的心脏损伤。Calpain 1和2(CPN1/2)是钙依赖的蛋白水解酶 在ISC-REP期间激活。CPN1/2的激活通过降解结构蛋白来损害心脏功能， 包括血影蛋白和连接素-2(JPH2)。CPN1/2的激活也是线粒体功能障碍的原因 在ISC代表期间。此外，CPN1/2的激活通过裂解和激活caspase-1来增加炎症。 因此，我们将测试给予CPN1/2抑制剂(MDL-28170，MDL)是否可以减少心脏损伤 在DCD的心中。我们将进一步测试CyA和MDL的联合治疗是否可以提供额外的保护 与单独使用CyA或MDL治疗DCD心脏相比，尤其是在缺血期更长的情况下。目标1 将评估大鼠DCD心脏的MPTP开放和CPN1/2激活，特别是较长时间的ISC。 我们将进一步研究长时间ISC的DCD心脏是否会进一步增加MPTP的开放 和CPN1/2的激活。目的2研究CyA或MDL治疗在减少心脏损伤方面的效果 在DCD心脏中单独和合并ISC的延长时期。目标3将研究保护 CyA或MDL对大鼠异位心脏移植的影响及较长时间的随访(2周)。至 建立我们工作的翻译潜力，我们将在DCD猪心汽车上测试CyA和MDL的好处- 移植模型。我们计划的实验和预期的结果将增加DCD心脏的可用性 对于HTX来说，即使ISC-Rep的周期更长。采用建议的综合治疗方法，我们的目标是 将DCD心脏用于临床移植。

Patients with end-stage heart failure (HF) require mechanical circulatory support, and if eligible, heart transplantation (HTx). The supply of donor hearts has reached a plateau since the only current source of donor hearts consists of patients with irreversible complete brain damage (donation after brain death, DBD). However, the number of eligible HT recipients increased exponentially. Unfortunately, due to this demand-supply mismatch, up to 20% of patients die while listed for HTx. Thus, there is an urgent need to expand the heart donor pool. A potential new source of such donor hearts is from DCD (donation after circulatory death) donors. DCD donors have increased the transplantation rates of solid organs, including liver, lungs and kidney, by 50%. Unfortunately, DCD protocol induces a sustained warm ischemic time that damages the myocardium precluding its use for clinical transplantation. Thus, the ischemia (ISC) from DCD protocol, the potential myocardial injury from storage, and reperfusion (REP) associated injury combine to represent additional risks to exacerbate injury in the DCD heart, precluding their routine use in clinical transplantation. A limited number of HTx are performed utilizing DCD hearts under strict protocol with very short ischemia times (<20 minutes). Extending the acceptable ischemia time (35 minutes) will allow significant additional DCD hearts to be utilized for HTx. Although the warm ISC is inevitable in DCD hearts, REP injury can be decreased through interventions applied at the onset of REP. We propose that the development of new strategies to prevent REP injury will reduce damage to the DCD heart. Mitochondria are critical targets and mediators of cardiac injury during REP. Mitochondrial permeability transition pore (MPTP) opening is considered a final step to induce cell death during ISC-REP. Since MPTP opening predominantly occurs during REP, there is a window of opportunity to decrease MPTP opening by applying intervention at the onset of REP. Cyclosporine A (CyA) is a classic MPTP inhibitor that decreases cardiac injury in hearts following ISC-REP. We propose that administration of the CyA at the onset of REP can reduce cardiac injury in the DCD hearts. Calpain1 and 2 (CPN1/2) are calcium-dependent proteases that are activated during ISC-REP. Activation of CPN1/2 impairs cardiac function by degrading structural proteins, including spectrin and junctophilin-2 (JPH2). Activation of CPN1/2 also contributes to mitochondrial dysfunction during ISC-REP. In addition, activation of CPN1/2 increases inflammation by cleaving and activating caspase-1. Therefore, we will test if the administration of CPN1/2 inhibitor (MDL-28170, MDL) can decrease cardiac injury in DCD hearts. We will further test if the combined treatment with CyA and MDL can provide additional protection compared to individual CyA or MDL treatment in DCD hearts, especially with a longer ischemia period. Aim 1 will evaluate the MPTP opening and CPN1/2 activation in rat DCD hearts, especially with a longer period of ISC. We will further study if REP after DCD hearts with a prolonged period of ISC further increases MPTP opening and CPN1/2 activation. Aim 2 will study the effectiveness of CyA or MDL treatment in decreasing cardiac injury in DCD hearts individually and in combination with a prolonged period of ISC. Aim 3 will study the protective effect of CyA or MDL in heterotopic transplanted rat hearts with a longer duration of follow-up (2 weeks). To establish the translation potential of our work, we will test the benefits of CyA and MDL in a DCD pig heart auto- transplantation model. Our planned experiments and the anticipated results will increase DCD hearts' availability for HTx even with longer periods of ISC-REP. Using an integrated treatment approach as proposed, we aim to move towards using DCD hearts for clinical transplantation.