The Mitochondrial Adenine Nucleotide Pool Contributes to the Matrix Ca2+ Buffering System
Authors: Davani Ariea J , Navarro Chris D , Heisner James S , Pannala Venkat R , Dash Ranjan K , Stowe David F , Camara Amadou KS 
Published: November 2023

Mitochondria have the ability to take up and store large amounts of Ca2+ to regulate and shape cytosolic Ca2+ transients. During cardiac ischemia (I) and reperfusion (R) injury, an overload of Ca2+ beyond the buffering capacity of mitochondria is implicated in causing deleterious consequences in cardiomyocytes; e.g. the opening of mitochondrial permeability transition pore (mPTP). Therefore, a better understanding of how to improve matrix Ca2+ buffering could represent a novel method to reduce damage induced during I/R injury. Adenine nucleotides (AdN) may have an effect on the buffering of matrix Ca2+. Thus, exploring the effect of altering matrix AdN on the buffering of matrix Ca2+ may reveal a potential method of mitigating excess Ca2+ loading during cardiac I/R injury. To explore this notion, mitochondria were isolated by differential centrifugation from guinea pig hearts. They were subsequently exposed to ADP, oligomycin (OMN; an inhibitor of mitochondrial ATP synthase), or the combination (OMN+ADP) to modify the matrix ATP/ADP ratio. The mitochondria were energized with pyruvic acid (PA) or succinic acid (SA). Next, the matrix Ca2+ retention capacity (CRC) was determined by exposing mitochondria to fixed CaCl2 boluses until mPTP opened (loss of matrix Ca2+). Ca2+ dynamics were measured inside (matrix) and outside (extra‐matrix) mitochondria. Membrane potential (ΔΨm) and NADH levels were also measured during CaCl2 bolus addition. Fluorescence spectrophotometry using specific dyes was used to assess Ca2+, NADH and ΔΨm. We found that adding ADP and OMN+ADP substantially increased the CRC and delayed mPTP opening for both substrates (SA and PA). During extra‐matrix Ca2+ measurements, addition of ADP alone increased the CRC by 200% and 300% (versus control) with PA and SA, respectively. Adding OMN+ADP increased the CRC by 212% and 257% for PA and SA, respectively. OMN alone increased the CRC by 50% for PA and 15% for SA. Similar patterns of CRCs were observed with matrix Ca2+ measurements for both PA and SA for all three treatment groups. The ΔΨm data revealed that addition of ADP resulted in 200% and 360% more Ca2+ to be added vs. control before maximal depolarization of ΔΨm for PA and SA, respectively. The OMN+ADP combination resulted in the addition of 300% and 320% more Ca2+ for PA and SA, respectively, before maximal ΔΨm depolarization. Adding OMN alone resulted in 20% more Ca2+ to be added vs. control before ΔΨm collapse for both substrates. The onset of NADH oxidation (loss of redox potential) reflected a similar pattern of increased Ca2+ buffering in the ADP and the OMN+ADP treatment groups. These results suggest that ADP alone and ADP+OMN enhance matrix Ca2+ buffering by altering the matrix ATP/ADP ratio. A change in the ATP/ADP ratio modifies mitochondrial CRC and prevents early mPTP opening in a substrate dependent manner. Alternatively, ADP and/or OMN potentially could modulate mPTP independently of matrix Ca2+ buffering. Enhancing matrix buffering of Ca2+ may facilitate maintenance of ΔΨm and redox state (NADH) and contribute to more Ca2+ uptake and buffering.Support or Funding InformationNIH grants T35 HL072483 and P01 GM066730