Dynamin-related protein 1 is a critical regulator of mitochondrial calcium homeostasis during myocardial ischemia/reperfusion injury

Abstract

Dynamin-related protein 1 (Drp1) is a cytosolic GTPase protein that when activated translocates to the mitochondria, meditating mitochondrial fission and increasing reactive oxygen species (ROS) in cardiomyocytes. Drp1 has shown promise as a therapeutic target for reducing cardiac ischemia/reperfusion (IR) injury; however, the lack of specificity of some small molecule Drp1 inhibitors and the reliance on the use of Drp1 haploinsufficient hearts from older mice have left the role of Drp1 in IR in question. Here, we address these concerns using two approaches, using: (a) short-term (3 weeks), conditional, cardiomyocyte-specific, Drp1 knockout (KO) and (b) a novel, highly specific Drp1 GTPase inhibitor, Drpitor1a. Short-term Drp1 KO mice exhibited preserved exercise capacity and cardiac contractility, and their isolated cardiac mitochondria demonstrated increased mitochondrial complex 1 activity, respiratory coupling, and calcium retention capacity compared to controls. When exposed to IR injury in a Langendorff perfusion system, Drp1 KO hearts had preserved contractility, decreased reactive oxygen species (ROS), enhanced mitochondrial calcium capacity, and increased resistance to mitochondrial permeability transition pore (MPTP) opening. Pharmacological inhibition of Drp1 with Drpitor1a following ischemia, but before reperfusion, was as protective as Drp1 KO for cardiac function and mitochondrial calcium homeostasis. In contrast to the benefits of short-term Drp1 inhibition, prolonged Drp1 ablation (6 weeks) resulted in cardiomyopathy. Drp1 KO hearts were also associated with decreased ryanodine receptor 2 (RyR2) protein expression and pharmacological inhibition of the RyR2 receptor decreased ROS in post-IR hearts suggesting that changes in RyR2 may have a role in Drp1 KO mediated cardioprotection. We conclude that Drp1-mediated increases in myocardial ROS production and impairment of mitochondrial calcium handling are key mechanisms of IR injury. Short-term inhibition of Drp1 is a promising strategy to limit early myocardial IR injury which is relevant for the therapy of acute myocardial infarction, cardiac arrest, and heart transplantation.

FIGURE 1

Three‐week cardiomyocyte‐specific Drp1 ablation does not affect cardiac function, exercise capacity, and mitochondrial morphology. (A) Immunoblotting of Drp1 protein demonstrates loss of Drp1 in the heart 3 weeks following tamoxifen injections. No Drp1 bands appear in the heart with Drp1 ablation. (B) The transverse section of the hearts. Trichrome staining on the heart tissue section shows mild signs of fibrosis in the heart with 3 weeks of Drp1 ablation. (C) M‐mode echocardiographic analysis shows no changes in fractional shorting (%) and heart weight with 3 weeks of Drp1 ablation. (D) The treadmill distance is not changed with 3 weeks of Drp1 ablation.

FIGURE 2

Three‐week Drp1 ablation improves ATP‐related oxygen consumption by increasing complex I activity. (A) Isolated mitochondria from control and Drp1 ablated hearts underwent analysis for oxygen consumption rates (OCR) using a XF24 Analyzer (Seahorse Bioscience). The sequential injection of mitochondrial inhibitors is indicated by arrows. (B) Mean values of baseline OCR, ATP‐related OCR, maximal OCR, State 3/State 4, and proton leak are compared between WT mitochondria and the mitochondria with 3‐week Drp1 ablation heart. (C) Complex I enzyme activity of isolated mitochondria from control hearts and Drp1 ablated hearts evaluated with commercial dipstick assay. (D) Complex I activity is increased in the Drp1 ablation heart by using a colorimetric assay to track the oxidation of NADH. (E) Complex III remains unchanged by using a colorimetric assay to monitor the reduction of cytochrome C.

FIGURE 3

Three‐week Drp1 ablation improves the hemodynamics and oxygen consumption following ischemia‐reperfusion (IR). (A) Representative traces of Langendorff perfused control and Drp1 ablated hearts following 30 min of ischemia followed by reperfusion. (B) Mean values of traces in A. (C) Oxygen consumption rate (OCR) traces determined by seahorse flux analysis on isolated mitochondria from control and Drp1 ablated hearts following IR injury. ADP addition was used to stimulate ATP‐coupled respiration. ATP synthase inhibitor oligomycin was used to block ATP‐coupled respiration, FCCP was used to uncouple mitochondrial respiration, and antimycin was used to inhibit mitochondrial respiration. The sequential injection of mitochondrial inhibitors is indicated by arrows. (D) Mean values of baseline OCR, ATP‐related OCR, maximal OCR, and State 3/State 4 are compared between WT mitochondria and the mitochondria in a 3‐week Drp1 knockout heart. (E) Complex I activity dipstick assay of mitochondrial complex I activity in control and Drp1 KO mitochondria following IR.

FIGURE 4

Three‐week Drp1 ablation rescues mitochondrial morphology and limits ROS generation in the heart post‐IR. (A) Representative transmission electron microscopy images of cardiac mitochondria and the mean values of mitochondria size and density in the WT group and Drp1 knockout group following IR. (B) MitoSox Red staining shows that mitochondrial ROS generation is normalized in the Drp1 knockout group post‐IR. (C) Succinate‐induced H₂O₂ production in WT cardiac mitochondria increases along with the increase of calcium concentration. Succinate‐induced H₂O₂ production in the Drp1 knockout group is insensitive to the increased calcium concentration. (D) Western blot bands and the bar graphs show that the increased cytochrome c expression is reduced in the Drp1KO group post‐IR.

FIGURE 5

Three‐week Drp1 ablation limits MPTP opening following IR injury. (A) Mitochondria isolated from control and Drp1 ablated hearts underwent calcium‐induced swelling assay to evaluate for MPTP opening. (B) Mitochondria from control and Drp1 ablated hearts following IR injury subjected to calcium‐induced swelling. (C) Mitochondria isolated from control and Drp1 ablated hearts subjected to calcium retention assay. (D) Mitochondria isolated from control and Drp1 ablated hearts following IR injury subjected to calcium retention assay. (E) mRNA expression of the RyR2 in control and Drp1 KO hearts at baseline and after IR injury. (F) Protein expression of the RyR2 in control and Drp1 KO hearts at baseline and after IR injury. (G) MitoSox staining of the left ventricle demonstrates decreased ROS generation in Langendorff hearts perfused with ruthenium red (RR).

FIGURE 6

Drp1 GTPase inhibitor Drpitor1a improves cardiac function by improving mitochondrial calcium homeostasis and reducing ROS generation. Representative pressure traces (A) and mean values (B) of Langendorff perfused hearts following IR with and without Drpitor1a. Following IR injury, diastolic pressure is elevated, and the systolic pressure is reduced resulting in a significantly decreased developed pressure (systolic pressure—end‐diastolic pressure). Administration of Drpitor1a following ischemia and at the beginning of reperfusion results in improved developed pressure. (C) Cardiac mitochondrial swelling (decrease in absorbance at 540 nm) induced by multiple injections of CaCl₂ shows MPTP opening was reduced by the treatment of Drpitor1a compared with the control group post‐IR. (D) Calcium retention capacity assay measured with the florescence density of calcium green‐5 N shows calcium uptake from the cytosol to mitochondria is increased with the treatment of Drpitor1a treatment compared with the control mitochondria post‐IR. (E) ROS indicator (MitoSox Red) staining of the left ventricle of IR hearts treated with and without Drpitor1a.

FIGURE 7

Proposed mechanism of acute Drp1 ablation on improving mitochondria and cardiac function post‐ischemia‐reperfusion (IR). In control of normal hearts Drp1 cycles on and off the outer mitochondrial membrane (OMM) maintaining mitochondrial homeostasis. IR injury results in increased cytosolic and mitochondrial matrix calcium. Drp1 translocated to the OMM resulting in increased mitochondrial permeability transition pore (MPTP) sensitivity resulting in membrane depolarization, mitochondrial swelling, and release of ROS and cytochrome C. In contrast, lack of Drp1 results in MPTP resistance to calcium overload and lack of pore opening resulting in maintained mitochondrial function. Lack of Drp1 also results in decreased expression of the cardiac calcium‐regulating ryanodine receptor (RyR2) protein which may also affect post‐IR calcium regulation.