Cyclophilin D-mediated regulation of the permeability transition pore is altered in mice lacking the mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU) is a multicomponent channel that is the primary mechanism for mitochondrial Ca 2+ uptake ( m Ca 2+ ). We previously reported that the MCU is required for energetic signaling to meet contractile demand during the ‘fight or flight’ response. In addition, we showed that deletion of the pore-forming component ( Mcu gene) protected against mitochondria permeability transition pore (MPTP) opening and ischemia-reperfusion injury. However, results from our study and others questioned the physiological relevance of MCU-mediated Ca 2+ uptake during chronic stress states featuring sustained intracellular Ca 2+ load ( i Ca 2+ ). To address this, we deleted Mcu from cardiomyocytes in adult mice ( Mcu cKO) and implanted osmotic pumps to deliver the β adrenergic agonist isoproterenol (iso, 70 mg/kg/day for 14d). In contrast to controls, Mcu cKO mice lacked contractile responsiveness to chronic βAR stimulation with evidence of LV dysfunction and failure by d14 ( Fig 1 ). Next, we crossed the Mcu cKO with mice overexpressing the β2a subunit (β2a-Tg) of the L-type Ca 2+ channel (LTCC). This model displays enhanced LTCC activity and cardiac contractility, but with added stress such as iso infusion, Ca 2+ overload eventually leads to MPTP-dependent cell death and heart failure. Surprisingly, loss of Mcu in this model was lethal with all mice dying by d13 ( Fig 2 ). Baseline echocardiography revealed that loss of Mcu ablated all β2a-mediated enhancements in LV contractility and accelerated dysfunction post-iso. These findings demonstrate that MCU-mediated m Ca 2+ uptake is critical to meet energetic demand during chronic stress states featuring sustained i Ca 2+ load.

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Abstract 506: Enhanced Mitochondrial Calcium Uptake During Chronic β-adrenergic Stimulation Causes Maladaptive Remodeling and Impaired Left Ventricular Function Independent of Cyclophilin D-mediated Mitochondrial Permeability Transition

Circulation Research ◽

10.1161/res.127.suppl_1.506 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Joanne F Garbincius ◽

Timothy S Luongo ◽

Jonathan P Lambert ◽

Adam S Mangold ◽

Emma K Murray ◽

...

Keyword(s):

Mitochondrial Permeability Transition ◽

Contractile Function ◽

Permeability Transition ◽

Mitochondrial Calcium ◽

Cyclophilin D ◽

Mitochondrial Calcium Uniporter ◽

Calcium Uniporter ◽

Isoproterenol Infusion ◽

Fractional Shortening

The mitochondrial calcium uniporter (MCU) forms the pore of the mitochondrial calcium uniporter channel (mtCU) and is required for rapid mitochondrial Ca 2+ ( m Ca 2+ ) uptake. MCU is necessary to increase cardiac energetics to fuel an increase in cardiac contractility during acute sympathetic stimulation. However, little is known about how MCU-dependent Ca 2+ flux may contribute to the heart’s adaptations to chronic stress. We therefore compared mice with adult cardiomyocyte (ACM)-specific loss ( Mcu fl/fl ; Mcu-cKO) or gain (CAG-CAT-MCU; MCU-Tg) of MCU function to examine the role of MCU-dependent m Ca 2+ uptake in a model of chronic catecholamine overload. In vitro characterization of ACMs confirmed that MCU overexpression enhanced and Mcu deletion inhibited acute m Ca 2+ uptake. Neither loss nor gain of MCU function altered baseline contractile function in vivo . In αMHC-MCM control mice, fractional shortening was transiently increased after 2 days of isoproterenol infusion. This initial increase in contractility was attenuated in MCU-cKO mice. In contrast, MCU-Tg mice exhibited decreased fractional shortening at 7 and 14 days of isoproterenol infusion. This detrimental effect on contractile function was associated with increased LV dilation, HW/BW ratio, and lung edema. MCU-Tg cardiomyocytes in vitro exhibited increased ROS production and a trend towards increased cell death upon elevation of cytosolic Ca 2+ with ionomycin. These data prompted us to test the hypothesis that isoproterenol-induced contractile dysfunction in MCU-Tg hearts is caused by cardiomyocyte dropout due to m Ca 2+ overload and mitochondrial permeability transition. However, genetic deletion of the mPTP component cyclophilin D did not prevent the decline in contractile function, diminish cardiomyocyte death, or attenuate LV remodeling in MCU-Tg animals during chronic isoproterenol infusion. We conclude that although mtCU-dependent m Ca 2+ uptake is essential for early energetic adaptations to high adrenergic load, under conditions of chronic adrenergic stress it is maladaptive and predisposes to heart failure. Our data suggest that this maladaptive response occurs via mechanisms independent of cyclophilin D-mediated permeability transition.

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HYPOTHYROIDISM MODULATES MITOCHONDRIAL CALCIUM UNIPORTER EXPRESSION AND CAUSES CARDIOPROTECTION DECRESING MITOCHONDRIAL PERMEABILITY TRANSITION PORE OPENING

Journal of the American College of Cardiology ◽

10.1016/s0735-1097(17)34144-x ◽

2017 ◽

Vol 69 (11) ◽

pp. 755

Author(s):

Hector Chapoy ◽

Yuriana Oropeza-Almazan ◽

Ana Gutiérrez-Rodríguez ◽

Gerardo Garcia-Rivas

Keyword(s):

Mitochondrial Permeability Transition Pore ◽

Mitochondrial Permeability Transition ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Mitochondrial Calcium ◽

Mitochondrial Calcium Uniporter ◽

Mitochondrial Permeability ◽

Calcium Uniporter ◽

Permeability Transition Pore Opening ◽

Pore Opening

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Abstract 92: The Mitochondrial Calcium Uniporter is Necessary for Metabolic Matching of Contractile Demand During Acute Sympathetic Stress

Circulation Research ◽

10.1161/res.117.suppl_1.92 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Timothy S Luongo ◽

Jonathan P Lambert ◽

Ancai Yuan ◽

Xueqian Zhang ◽

Santhanam Shanmughapriya ◽

...

Keyword(s):

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Mitochondrial Calcium ◽

Mitochondrial Calcium Uniporter ◽

Adult Mice ◽

Calcium Uniporter ◽

Myocardial Ischemia Reperfusion ◽

Sympathetic Stress

Contractility is mediated by a variable flux in intracellular calcium (Ca 2+ ), which is proposed to be integrated into mitochondria to regulate cardiac energetics. Moreover, m Ca 2+ -overload is known to activate the mitochondrial permeability transition pore (MPTP) and induce cell death. Recent studies have reported that the Mcu gene encodes the channel-forming portion of the mitochondrial calcium uniporter (MCU) and is required for m Ca 2+ uptake. To examine the role of m Ca 2+ in the heart, we generated a conditional, cardiac-specific knockout model and deleted Mcu in adult mice ( Mcu-cKO ). Loss of Mcu protected against myocardial ischemia-reperfusion (IR) injury by preventing the activation of the MPTP. In addition while we found no baseline phenotype, Mcu -cKO mice lacked contractile responsiveness to beta-adrenergic receptor stimulation as assessed by invasive hemodynamics (Fig 1) and in parallel were unable to activate mitochondrial dehydrogenases and increase cardiac NADH levels. Further experimental analyses in isolated adult cardiomyocytes confirmed a lack of energetic responsiveness to acute sympathetic stress (isoproterenol failure to mediate an increase in NADH, Fig 2), supporting the hypothesis that the physiological function of the MCU in the heart is to modulate Ca 2+ -dependent metabolism during the ‘fight or flight’ response.

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Deletion of mitochondrial calcium uniporter incompletely inhibits calcium uptake and induction of the permeability transition pore in brain mitochondria

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.002926 ◽

2018 ◽

Vol 293 (40) ◽

pp. 15652-15663 ◽

Cited By ~ 13

Author(s):

James Hamilton ◽

Tatiana Brustovetsky ◽

Jacob E. Rysted ◽

Zhihong Lin ◽

Yuriy M. Usachev ◽

...

Keyword(s):

Calcium Uptake ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Brain Mitochondria ◽

Mitochondrial Calcium ◽

Mitochondrial Calcium Uniporter ◽

Calcium Uniporter

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Abstract MP124: De-palmitoylation of Cysteine 202 of Cyclophilin-D by Calcium is Important for the Activation of the Permeability Transition Pore

Circulation Research ◽

10.1161/res.127.suppl_1.mp124 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Georgios Amanakis ◽

Junhui Sun ◽

Maria Fergusson ◽

Chengyu Liu ◽

Jeff D Molkentin ◽

...

Keyword(s):

Oxidative Stress ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Calcium Overload ◽

Mitochondrial Calcium ◽

Cyclophilin D ◽

Perfused Heart ◽

Knock Out

Cyclophilin-D (CypD) is a well-known regulator of the mitochondrial permeability transition pore (PTP), the main effector of cardiac ischemia/reperfusion (I/R) injury characterized by oxidative stress and calcium overload. However, the mechanism by which CypD activates PTP is poorly understood. Cysteine 202 of CypD (C202) is highly conserved across species and can undergo redox-sensitive post-translational modifications, such as S-nitrosylation and oxidation. To study the importance of C202, we developed a knock-in mouse model using CRISPR where CypD-C202 was mutated to a serine (C202S). Hearts from these mice are protected against I/R injury. We found C202 to be abundantly S-palmitoylated under baseline conditions while C202 was de-palmitoylated during ischemia in WT hearts. To further investigate the mechanism of de-palmitoylation during ischemia, we considered the increase of matrix calcium, oxidative stress and uncoupling of ATP synthesis from the electron transport chain. We tested the effects of these conditions on the palmitoylation of CypD in isolated cardiac mitochondria. The palmitoylation of CypD was assessed using a resin-assisted capture (Acyl-RAC). We report that oxidative stress (phenylarsenide) and uncoupling (CCCP) had no effect on CypD palmitoylation (p>0.05, n=3 and n=7 respectively). However, calcium overload led to de-palmitoylation of CypD to the level observed at the end ischemia (1±0.10 vs 0.63±0.09, p=0.012, n=9). To further test the hypothesis that calcium regulates S-palmitoylation of CypD we measured S-palmitoylation of CypD in non-perfused heart lysates from global germline mitochondrial calcium uniporter knock-out mice (MCU-KO), which have reduced mitochondrial calcium and we found an increase in S-palmitoylation of CypD (WT 1±0.04 vs MCU-KO 1.603±0.11, p<0.001, n=6). The data are consistent with the hypothesis that C202 is important for the CypD mediated activation of PTP. Ischemia leads to increased matrix calcium which in turn promotes the de-palmitoylation of CypD on C202. The now free C202 can further be oxidized during reperfusion leading to the activation of PTP. Thus, S-palmitoylation and oxidation of CypD-C202 possibly target CypD to the PTP, making them potent regulators of cardiac I/R injury.

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