Dynamin-related protein 1 (Drp1)-mediated diastolic dysfunction in myocardial ischemia-reperfusion injury: therapeutic benefits of Drp1 inhibition to reduce mitochondrial fission

Abstract

Mitochondrial fission, regulated by dynamin-related protein-1 (Drp1), is a newly recognized determinant of mitochondrial function, but its contribution to left ventricular (LV) impairment following ischemia-reperfusion (IR) injury is unknown. We report that Drp1 activation during IR results in LV dysfunction and that Drp1 inhibition is beneficial. In both isolated neonatal murine cardiomyocytes and adult rat hearts (Langendorff preparation) mitochondrial fragmentation and swelling occurred within 30 min of IR. Drp1-S637 (serine 637) dephosphorylation resulted in Drp1 mitochondrial translocation and increased mitochondrial fission. The Drp1 inhibitor Mdivi-1 preserved mitochondrial morphology, reduced cytosolic calcium, and prevented cell death. Drp1 siRNA similarly preserved mitochondrial morphology. In Langendorff hearts, Mdivi-1 reduced mitochondrial reactive oxygen species, improved LV developed pressure (92±5 vs. 28±10 mmHg, P<0.001), and lowered LV end diastolic pressure (10±1 vs. 86±13 mmHg, P<0.001) following IR. Mdivi-1 was protective if administered prior to or following ischemia. Because Drp1-S637 dephosphorylation is calcineurin sensitive, we assessed the effects of a calcineurin inhibitor, FK506. FK506 treatment prior to IR prevented Drp1-S637 dephosphorylation and preserved cardiac function. Likewise, therapeutic hypothermia (30°C) inhibited Drp1-S637 dephosphorylation and preserved mitochondrial morphology and myocardial function. Drp1 inhibition is a novel strategy to improve myocardial function following IR.

Keywords: IR; Mdivi-1; calcineurin; cardiac arrest; reactive oxygen species.

Figure 1.

Drp1 mediates IR-induced mitochondrial fission. A) Isolated neonatal murine ventricular myocytes were infected with BacMam mito-GFP for 24–48 h and exposed to 30 min of IR (note mitochondrial fragmentation and swelling). Inhibiting Drp1 (using Mdivi-1 or siDrp1) or TH decreased mitochondrial fragmentation, quantified as a decrease in MFC. n ≥ 13/group. Scale bar = 10 μM. B) Representative TEM images and mean values for mitochondrial area in Langendorff hearts exposed to 30 min of IR. IR causes significant mitochondrial swelling that Mdivi-1 or TH prevents. n ≥ 300/group. Scale bar = 1 μM.

Figure 2.

Drp1 inhibition during IR preserves cardiomyocyte function. A) IR-induced cardiomyocyte cell death was inhibited by Mdivi-1 and TH. n = 5. B) IR-induced elevations of cytosolic calcium were prevented by inhibition of Drp1 (siDrp1 and Mdivi-1) and TH, but not by vehicle or SCR siRNA controls in isolated cardiomyocytes. n ≥ 21. C) Drp1 inhibition with Mdivi-1 prevented decreases in OCR in isolated cardiomyocytes by IR. n ≥ 12. D) Tissue ATP concentrations decreased following IR and were partially preserved by TH, but not Mdivi-1. n = 5. E) IR-induced increases in mitochondrial-derived ROS (MitoSOX) were inhibited by Mdivi-1 and TH. n ≥ 9/group. Differential interference microscopy (DIC) images are shown below their respective MitoSOX images. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Strategies to inhibit dephosphorylation of Drp1 S637 and mitochondrial Drp1 translocation. IR-induced dephosphorylation of Drp1 S637 and mitochondrial translocation were prevented by Mdivi-1 and TH (A, B) as well as FK506 (C, D) in whole-cell lysates (A, C) and mitochondrial fraction B, D).

Figure 4.

Drp1 inhibition improves diastolic function following IR. Representative traces (A) and mean data (B) show that, after 30 min of IR, developed pressure (systolic-diastolic pressure) was severely reduced, and diastolic pressure was dramatically elevated. Pretreatment with Mdivi-1, TH, or FK506 significantly improved diastolic function following IR. n = 7/group.

Figure 5.

Postischemic administration of Mdivi-1 improves diastolic function. Isolated Langendorff hearts underwent 20 min of perfusion, 30 min of ischemia, and an additional 20 min of reperfusion. Mdivi-1 (25 μM) was administered at the onset of reperfusion. A) Mdivi-1 postischemic administration preserved relatively lower DP. B) Pulse pressure was significantly improved compared to IR control (DMSO). n = 7.

Figure 6.

Mechanistic pathway of Drp1 activation illustrating how Drp1 may induce contractile dysfunction following IR.