Regulation of the Human Cardiac Mitochondrial Ca ²⁺ Uptake by 2 Different Voltage-Gated Ca ²⁺ Channels

Abstract

Background— Impairment of intracellular Ca 2+ homeostasis and mitochondrial function has been implicated in the development of cardiomyopathy. Mitochondrial Ca 2+ uptake is thought to be mediated by the Ca 2+ uniporter (MCU) and a thus far speculative non-MCU pathway. However, the identity and properties of these pathways are a matter of intense debate, and possible functional alterations in diseased states have remained elusive. Methods and Results— By patch clamping the inner membrane of mitochondria from nonfailing and failing human hearts, we have identified 2 previously unknown Ca 2+ -selective channels, referred to as mCa1 and mCa2. Both channels are voltage dependent but differ significantly in gating parameters. Compared with mCa2 channels, mCa1 channels exhibit a higher single-channel amplitude, shorter openings, a lower open probability, and 3 to 5 subconductance states. Similar to the MCU, mCa1 is inhibited by 200 nmol/L ruthenium 360, whereas mCa2 is insensitive to 200 nmol/L ruthenium 360 and reduced only by very high concentrations (10 μmol/L). Both mitochondrial Ca 2+ channels are unaffected by blockers of other possibly Ca 2+ -conducting mitochondrial pores but were activated by spermine (1 mmol/L). Notably, activity of mCa1 and mCa2 channels is decreased in failing compared with nonfailing heart conditions, making them less effective for Ca 2+ uptake and likely Ca 2+ -induced metabolism. Conclusions— Thus, we conclude that the human mitochondrial Ca 2+ uptake is mediated by these 2 distinct Ca 2+ channels, which are functionally impaired in heart failure. Current properties reveal that the mCa1 channel underlies the human MCU and that the mCa2 channel is responsible for the ruthenium red–insensitive/low-sensitivity non-MCU–type mitochondrial Ca 2+ uptake.

RETRACTED: Glycogen synthase kinase 3β transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K + channels

“…Freshly isolated cardiomyocytes were used within 1-2 h. Cardiomyocytes were labeled with 1 μM Mitotracker Green (Molecular Probes, Inc.) to facilitate identification of intact mitoplasts after further subcellular purification. Isolated intact subsarcolemmal and interfibrillar mitoplasts were prepared as previously reported (9,38,39). Mitochondria were stored at 4°C for up to 48 h for patch-clamp experiments.…”

“…All experiments were performed in the mitoplastattached configuration of the patch-clamp technique (180 test pulses of 150-ms duration at 1.67 Hz, sampling frequency of 10 kHz, corner frequency of 2 kHz) with symmetrical bath and pipette solution composed of 150 mM KCl and 10 mM K-Hepes, with pH adjusted to 7.2 with potassium hydroxyde (KOH), as previously described (9,38,39). Currents were recorded and digitized with an Axopatch 200B amplifier and Digidata 1200 interface (Axon Instruments).…”

“…Currents were recorded and digitized with an Axopatch 200B amplifier and Digidata 1200 interface (Axon Instruments). Single-channel analysis was done using custom software only from one-channel patches as previously reported (9,38,39). For detailed gating analysis, idealized currents were analyzed in 150-ms steps (if not indicated otherwise).…”

Mitochondrial Ca 2+ Influx Contributes to Arrhythmic Risk in Nonischemic Cardiomyopathy

“…Consistent with the latter 2 possibilities, we observed increased whole‐mitoplast MCU current. Our result differs from that of Michels et al, who reported a decrease in mitochondrial Ca 2+ uptake in a heterogeneous group of human failing hearts 48. The reason for this difference is not clear, but our results clearly show that mitochondrial Ca 2+ handling can contribute to EADs and arrhythmia in chronic and moderate HF.…”

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

“…However, in heart failure, it has been well reported that there is a significant elevation of cytosolic Na 2+ concentration ( addition, the [Na 2+ ] c elevation and mitochondrial NCX activity in heart failure dictate mROS generation through a decrease in the mitochondrial Ca 2+ uptake (50). Indeed, several studies showed that mitochondrial Ca 2+ uptake is reduced in failing cardiomyocytes (43,47). These reports demonstrated that elevated [Na + ] c and mitochondrial NCX activity also critically contribute to decreased [Ca 2+ ] mt , especially during heart failure.…”