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，心脏

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.