Mitochondrial Ca2+ Influx Contributes to Arrhythmic Risk in Nonischemic Cardiomyopathy

Abstract

Background: Heart failure (HF) is associated with increased arrhythmia risk and triggered activity. Abnormal Ca²⁺ handling is thought to underlie triggered activity, and mitochondria participate in Ca²⁺ homeostasis.

Methods and results: A model of nonischemic HF was induced in C57BL/6 mice by hypertension. Computer simulations were performed using a mouse ventricular myocyte model of HF. Isoproterenol-induced premature ventricular contractions and ventricular fibrillation were more prevalent in nonischemic HF mice than sham controls. Isolated myopathic myocytes showed decreased cytoplasmic Ca²⁺ transients, increased mitochondrial Ca²⁺ transients, and increased action potential duration at 90% repolarization. The alteration of action potential duration at 90% repolarization was consistent with in vivo corrected QT prolongation and could be explained by augmented L-type Ca²⁺ currents, increased Na⁺-Ca²⁺ exchange currents, and decreased total K⁺ currents. Of myopathic ventricular myocytes, 66% showed early afterdepolarizations (EADs) compared with 17% of sham myocytes (P<0.05). Intracellular application of 1 μmol/L Ru360, a mitochondrial Ca²⁺ uniporter-specific antagonist, could reduce mitochondrial Ca²⁺ transients, decrease action potential duration at 90% repolarization, and ameliorate EADs. Furthermore, genetic knockdown of mitochondrial Ca²⁺ uniporters inhibited mitochondrial Ca²⁺ uptake, reduced Na⁺-Ca²⁺ exchange currents, decreased action potential duration at 90% repolarization, suppressed EADs, and reduced ventricular fibrillation in nonischemic HF mice. Computer simulations showed that EADs promoted by HF remodeling could be abolished by blocking either the mitochondrial Ca²⁺ uniporter or the L-type Ca²⁺ current, consistent with the experimental observations.

Conclusions: Mitochondrial Ca²⁺ handling plays an important role in EADs seen with nonischemic cardiomyopathy and may represent a therapeutic target to reduce arrhythmic risk in this condition.

Keywords: arrhythmia; calcium; heart failure; mitochondria.

Figure 1

Computer model of mouse ventricular cell. A, Schematic diagrams of the 3‐dimensional structure of the cell model (left) and the Ca²⁺ release unit (CRU)–mitochondrial Ca²⁺ cycling model (right). B, The modified L‐type Ca²⁺ current model. Left: The Hodgkin‐Huxley (HH) scheme. Right: The equivalent Markov scheme of the HH scheme. To simulate a much lower channel open probability (≈5%–10%) observed in experiments, we added a new state (the final open state), with the opening rate from the d₂f₂f_Ca2 state being r₁ and the closing rate being r₂. CYTO indicates cytosolic space; DS, dyadic space; jm‐NaCa, mitochondrial Na⁺‐Ca²⁺ exchanger Ca2+ release; JSR, junctional sarcoplasmic reticulum; j_uni, mitochondrial Ca²⁺ uniporter uptake; j_up, sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) uptake; LCC, L‐type Ca²⁺ channel; MITO, mitochondrial space; NSR, network sarcoplasmic reticulum; RyR, ryanodine receptor; SUB, submembrane space.

Figure 2

Telemetry of sham and nonischemic heart failure (NI‐HF) C57BL/6 mice. A, Examples of telemetric ECG recordings. ECG signals in the left and right panels were sampled from sham and NI‐HF mice, respectively. Waveforms were collected before (control) and after (0.2 and 2.5 mg/kg IP) isoproterenol injection. B, Heart rate (HR; beats per minute) and corrected QT (QTc) intervals (ms) were measured from lead II and plotted in left and right panels, respectively. Compared with the control mice, NI‐HF mice showed a longer QT interval. n=7 (mice) for each group. *P<0.05 compared with that in sham group.

Figure 3

Representative action potentials (APs), mitochondrial Ca²⁺ transients, mitochondrial Ca²⁺ uniporter current (I_MCU), and cytosol Ca²⁺ transients recorded from sham and nonischemic heart failure (NI‐HF) mouse ventricular myocytes. A, APs and mitochondrial Ca²⁺ transients simultaneously recorded from cardiomyopathic ventricular cells showing early afterdepolarizations (EADs). Stimulation (0.5 Hz) was used to evoke APs. The time scale bar is shown at 0 mV. Mitochondrial Ca²⁺ oscillations corresponded with EADs. B, Left panel: I_MCU in mitoplasts isolated from both sham and NI‐HF ventricular cardiomyocytes. Right panel: The average I_MCU at −160 mV (n=8 for sham, and n=7 for NI‐HF). C, Cytoplasmic Ca²⁺ transients without EADs. F/F₀ indicates background‐subtracted normalized fluorescence.

Figure 4

Typical action potential (AP) and mitochondrial Ca²⁺ traces recorded synchronously from nonischemic heart failure (NI‐HF) mice and NI‐HF mice treated with Ru360 (1 µmol/L). Top panel: APs. Bottom panel: Mitochondrial Ca²⁺ transients. Triggered activity was inhibited by intracellular application of 1 µmol/L Ru360 in NI‐HF cardiomyocytes. Stimulation (0.5 Hz) was used to evoke APs. The time scale bar is shown at 0 mV. F/F₀ indicates background‐subtracted normalized fluorescence.

Figure 5

Comparison of membrane currents in sham and nonischemic heart failure (NI‐HF) cardiomyocytes. A, Top panel: K⁺ currents recorded at +50 mV from both sham and NI‐HF cardiomyocytes. Middle panel: The average peak amplitudes of total K⁺ currents (I_K ⁺ _‐peak) are shown (n=4 cells from 2 sham mice, and n=3 cells from 1 NI‐HF mouse). The holding potential was −80 mV. Voltages were from −110 to +50 mV, with a step of +10 mV and a duration of 5 seconds. Bottom panel: The average steady‐state currents of total K⁺ currents (I_K ⁺ _{‐ SS}) are drawn (n=4 cells from 2 sham mice, and n=3 cells from 1 NI‐HF mouse). B, Top panel: L‐type Ca²⁺ currents recorded at 0 mV from both sham and NI‐HF cardiomyocytes. Bottom panel: The average peak amplitudes of L‐type Ca²⁺ currents (n=4 cells from 1 sham mouse, and n=5 cells from 1 NI‐HF mouse). The holding potential was −50 mV. Voltages were from −40 to +60 mV, with a step of +10 mV and a duration of 300 ms. C, The average inward Na⁺‐Ca²⁺ exchanger (NCX) currents (n=7 cells from 2 mice in sham group, and n=9 cells from 2 mice in NI‐HF group). Cells were put in a solution containing no Na⁺ and 10 µmol/L ryanodine. The holding potential was −40 mV. Ten 100‐ms steps from −40 to +10 mV were followed by a step to −100 mV to repolarized cells. This step was accompanied by rapidly applied Na⁺ to record NCX currents. *P<0.05 compared with that in sham group.

Figure 6

Mitochondrial Ca²⁺ uniporter (MCU) expression in wild‐type (WT) and MCU ^+/− mice and Na⁺‐Ca²⁺ exchanger (NCX) currents of WT nonischemic heart failure (NI‐HF) and MCU ^+/− NI‐HF mice. A, Representative Western blot results: Compared with WT mice, the MCU protein level is decreased in MCU ^+/− mice hearts. B, MCU mRNA expression obtained by quantitative real‐time reverse transcription–polymerase chain reaction is substantially reduced in MCU ^+/− mice hearts by 58% (n=6 mice in WT group, and n=8 mice in MCU ^+/− group). C, Compared with NI‐HF mice ventricular cardiomyocytes, the membrane NCX currents are significantly decreased in MCU ^+/− NI‐HF mice cardiomyocytes (n=7 cells from 2 mice in NI‐HF cardiomyocytes, and n=6 cells from 2 mice in MCU ^+/− NI‐HF cardiomyocytes). *P<0.05, **P<0.01 compared with that in NI‐HF group.

Figure 7

Action potentials (APs) and mitochondrial Ca²⁺ traces recorded simultaneously from nonischemic heart failure (NI‐HF) and mitochondrial Ca²⁺ uniporter (MCU) ^+/− NI‐HF mice. Top panel: APs. Early afterdepolarizations were eliminated in MCU ^+/− NI‐HF mouse cardiomyocytes. Bottom panel: Mitochondrial Ca²⁺ transients. Stimulation (0.5 Hz) was used to evoke APs. The time scale bar is shown at 0 mV. F/F₀ indicates background‐subtracted normalized fluorescence.

Figure 8

Telemetry of nonischemic heart failure (NI‐HF) and mitochondrial Ca²⁺ uniporter (MCU)^+/− NI‐HF CD1 mice. A, Examples of telemetric ECG recordings. ECG signals in top and bottom panels were sampled from NI‐HF (n=5) and MCU ^+/− NI‐HF (n=6) mice, respectively. Waveforms were collected before (control) and after (IP 2.5 mg/kg) isoproterenol injection. B, Heart rate (HR) and corrected QT (QTc) were measured from lead II and plotted in left and right panels, respectively. Compared with the NI‐HF mice, MCU ^+/− NI‐HF mice showed a reduced QT interval (n=4 mice for the NI‐HF group, and n=5 mice for the MCU ^+/− NI‐HF group). *P<0.05 compared with that in NI‐HF group.

Figure 9

The alterations of mitochondrial properties in nonischemic heart failure (NI‐HF) mice. A, Fluorescence staining results revealed that the overall mitochondrial mass increased from background‐subtracted normalized fluorescence (F/F_o) of 1.13±0.02 in sham ventricular cardiomyocytes (n=16) to 1.20±0.03 in NI‐HF cardiomyocytes (n=19). B, Compared with sham cardiomyocytes (n=12), the mitochondrial membrane potential was slightly depolarized in NI‐HF cardiomyocytes (n=10). Mitochondrial membrane potential was collapsed by 20 µmol/L carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP) in both sham and NI‐HF cardiomyocytes. C, Western blots of mitochondrial Ca²⁺ uniporter (MCU), mitochondrial Na⁺‐Ca²⁺ exchange (NCLX), and phosphorylated MCU. D, The expression of phosphorylated MCU was upregulated from 0.30±0.04 in sham group to 1.45±0.46 in NI‐HF group. GFP indicates green fluorescent protein; p‐tyrosine, phosphorylated tyrosine; TMRM, tetramethylrhodamine methyl ester. *P<0.05 compared with that in sham group.

Figure 10

Computer simulation results. A, Action potential duration (APD) and Ca²⁺ cycling dynamics under the normal condition. Left panel: Time traces of membrane voltage, cytosolic Ca²⁺ concentration, sarcoplasmic reticulum Ca²⁺ concentration, and mitochondrial free Ca²⁺ concentration under the normal control condition. Right panel: Dependence of the APD on mitochondrial Ca²⁺ uniporter (MCU) activity (α_MCU) and L‐type Ca²⁺ channel (LCC) conductance (α_gCaL). B, APD and Ca²⁺ cycling dynamics under the heart failure condition. The layout of time traces for each case is the same as in A. Top left: Heart failure controls with early afterdepolarizations (EADs). Top right: MCU activity was reduced by 3‐fold (α_MCU changed from 4 to 1). Bottom left: LCC conductance was reduced by 20% (α_gCaL changed from 1.1 to 0.9). Bottom right: Dependence of the APD on MCU activity and LCC conductance. In this map, the APD jumps suddenly from ≈50 to >300 ms when EADs occur. The 3 specific cases shown above were marked on the map with different symbols. I_Ca,L indicates L‐type Ca²⁺ current.

Figure 11

Mechanistic insights from the computer model into the roles of mitochondrial Ca²⁺ handling in the genesis of early afterdepolarizations (EADs). A through C, Ca²⁺ concentrations in different compartments of the cell model vs the strength of mitochondrial Ca²⁺ uniporter (MCU), showing bistability. The simulations were performed as follows. We first started the simulations from the normal control MCU activity (α_MCU=1) and increased α_MCU gradually to 35, as indicated by the blue arrows (B). The system switches from no EADs (also low Ca²⁺ concentration states) to EADs (also high Ca²⁺ concentration states) at α_MCU≈30, at which the mitochondrial free Ca²⁺ reaches 136 nmol/L (open arrow). For each α_MCU, 50 beats were simulated for the cell to reach steady state before changing to a larger α_MCU. We then started the simulations in the same way but from a high MCU activity (α_MCU=35) to the normal control value (α_MCU=1), as indicated by the red arrow (B). The system switches from EADs to no EADs at α_MCU≈2.4. D. [Ca²⁺]_mito, [Ca²⁺]_i, [Ca²⁺]_SR, I_NCX, I_{C a,L}, and V vs time from 2 simulations for α_MCU=5. In the first simulation (blue traces), the mitochondrial Ca²⁺ is free running (unclamped), and the cell is in the low Ca²⁺ state without EADs. In the second simulation (red traces), [Ca²⁺]_mito is suddenly elevated to 200 nmol/L and held constant (clamped) for the rest of the simulation. 1, 2, and 3 mark the first 3 beats during the clamped phase. [Ca²⁺]_mito indicates mitochondrial Ca²⁺ concentration; [Ca²⁺]_i, intracellular Ca²⁺ concentration; [Ca²⁺]_SR, SR Ca²⁺ concentration; INCX, NCX current; I_caL, L‐type Ca²⁺ current; V, membrane potential.

Figure 12

Schematic plot of the feedback loops involved in the action potential (AP) and Ca²⁺ cycling dynamics. APD indicates AP duration; [Ca²⁺]_mito, mitochondrial Ca²⁺ concentration; [Ca²⁺]_i, intracellular Ca²⁺ concentration; [Ca²⁺]_SR, SR Ca²⁺ concentration;EAD, early afterdepolarization; LCC, L‐type Ca²⁺ channel; MCU, mitochondrial Ca²⁺ uniporter; NCX, Na⁺‐Ca²⁺ exchange; RyR, ryanodine receptor; SERCA, sarco/endoplasmic reticulum Ca²⁺‐ATPase.