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Introduction Describes the unique properties of the pulmonary circulation and its response to low oxygen tension, known as hypoxic pulmonary vasoconstriction (HPV). Presents the role of smooth muscle cell contraction in vasoconstriction and the lack of a definitive mechanism for oxygen sensing in these cells. Discusses the role of mitochondria as a putative oxygen sensor and the potential regulation of Kv channels by mitochondrial factors. Highlights previous studies showing the effects of mitochondrial inhibitors and products on Kv channels in pulmonary arterial smooth muscle cells (PASMCs). Proposes that differences in mitochondrial localization in PASMCs and systemic mesenteric arterial smooth muscle cells (MASMCs) may contribute to the divergent oxygen sensitivity in the two circulations.

Materials and Methods Lists the materials used in the study, including chemicals, enzymes, and fluorescent probes. Describes the methods for cell isolation from rat small intrapulmonary arteries and mesenteric arteries. Details the confocal microscopy protocol for visualizing the spatial distribution of mitochondria and the sarcoplasmic reticulum (SR) in PASMCs and MASMCs. Explains the electrophysiology techniques used to record and analyze Kv currents (IKv) in both cell types. Outlines the data analysis and statistical methods employed in the study.

Results Presents confocal microscopy data demonstrating that mitochondria are located significantly closer to the plasmalemmal membrane in PASMCs compared with MASMCs. Shows that mitochondrial inhibitors antimycin A and oligomycin A have a more potent effect on IKv in PASMCs than in MASMCs, consistent with the differences in mitochondrial localization. Demonstrates that the cytoskeletal disruptor cytochalasin B alters mitochondrial distribution in PASMCs and attenuates the effect of antimycin A on IKv. Provides evidence that intracellular Mg2 is involved in antimycin A-mediated changes in IKv inactivation.

Discussion Discusses the implications of the subplasmalemmal localization of mitochondria in PASMCs for functional interaction with Kv channels. Proposes that the close proximity of mitochondria to the plasma membrane in PASMCs may contribute to the oxygen sensitivity of IKv in the pulmonary circulation. Explores the mechanisms involved in Kv channel regulation by mitochondria in PASMCs, highlighting the role of intracellular Mg2. Compares the functional coupling between mitochondria and Kv currents in PASMCs and MASMCs, emphasizing the stronger coupling in PASMCs. Discusses the distinct differences in Kv channel activity between PASMCs and MASMCs, suggesting potential contributions from channel subunit composition and expression levels. Concludes that the unique spatial organization of mitochondria in PASMCs, coupled with the differences in Kv channel properties, may underlie the distinct oxygen sensitivity of the pulmonary circulation.

The paper argues that in pulmonary artery smooth muscle cells (PASMCs), the mitochondria sit close to the plasma membrane and interact with voltage-gated potassium (Kv) channels. This unique spatial arrangement allows mitochondria to potentially act as oxygen sensors in these cells, which is particularly important in the pulmonary circulation.

Why It Matters

1. Hypoxic Pulmonary Vasoconstriction (HPV): HPV is a critical response in the lungs. When oxygen levels drop, pulmonary arteries constrict to redirect blood to better-ventilated areas, optimizing oxygen uptake. This response depends on PASMC contraction, but how these cells sense oxygen remains unclear.

2. Mitochondria as Oxygen Sensors: Since mitochondria consume oxygen to produce energy, they’re naturally sensitive to oxygen levels. The idea here is that mitochondria can relay information about oxygen availability directly to the Kv channels in the nearby plasma membrane, possibly modulating their activity. In low oxygen conditions (hypoxia), this interaction could trigger the changes in ion flow that lead to PASMC contraction, initiating HPV.

3. Kv Channels and Contraction: Kv channels help maintain membrane potential and influence calcium levels in cells. When they close (often due to low oxygen levels or mitochondrial signals), PASMCs depolarize, calcium channels open, and calcium floods in. This increase in intracellular calcium triggers contraction of the smooth muscle, leading to vasoconstriction. By understanding this mechanism, scientists could more accurately predict or manipulate HPV.

Why This Matters Clinically

• Pulmonary Hypertension and Disease Implications: Disruptions in oxygen sensing can lead to chronic HPV, contributing to pulmonary hypertension and heart failure. If mitochondria play a significant role in this oxygen sensing and Kv channel regulation, understanding this pathway could help in designing therapies that target mitochondrial function or Kv channels to treat diseases like pulmonary hypertension.

• Targeted Therapies: If we know mitochondria-Kv channel interactions are crucial for HPV, drugs or interventions could be developed to modulate this pathway, potentially providing new ways to control or prevent diseases where HPV is either excessive or insufficient.

Abstract

This study investigates the role of mitochondria in hypoxic pulmonary vasoconstriction ( HPV ) , focusing on how mitochondrial localization within pulmonary arterial smooth muscle cells ( PASMCs ) affects the function of voltage-gated K⁺ ( Kv ) channels , which are crucial in regulating cellular excitability. The researchers hypothesized that differences in mitochondrial distribution between PASMCs and systemic mesenteric arterial smooth muscle cells ( MASMCs ) contribute to the distinct oxygen sensitivities of these tissues.

Using immunofluorescent labeling and confocal microscopy , they compared the cellular localization of mitochondria in PASMCs and MASMCs. They found that mitochondria are significantly closer to the plasma membrane in PASMCs than in MASMCs. Functional coupling between mitochondria and Kv channels was assessed using the patch-clamp technique alongside mitochondrial inhibitors antimycin A ( complex III inhibitor ) and oligomycin A ( ATP synthase inhibitor ). The inhibitors had a more pronounced effect on Kv currents ( $I_{K_v}$$I_{K_v}$ ) in PASMCs compared to MASMCs.

Additionally , treatment with cytochalasin B , a cytoskeletal disruptor , altered mitochondrial distribution in PASMCs and attenuated the effect of antimycin A on the voltage-dependent properties of $I_{K_v}$$I_{K_v}$

These findings suggest that the closer proximity of mitochondria to the plasma membrane in PASMCs facilitates a stronger structural and functional coupling with Kv channels. This coupling likely plays a significant role in regulating pulmonary artery excitability during HPV.

Results

Differences in Spatial Distribution of Mitochondria:

• Immunofluorescent Imaging: Confocal microscopy revealed that in PASMCs , mitochondria are predominantly located near the plasma membrane , appearing before the sarcoplasmic reticulum ( SR ) in optical sections moving from the cell bottom upward. In contrast , in MASMCs , the SR appeared before or simultaneously with mitochondria , indicating that mitochondria are situated deeper within the cell.

• Quantitative Analysis: Measurement of the distance between peripheral mitochondria and the plasma membrane showed that mitochondria in PASMCs are on average 1.2 μm away from the membrane , while in MASMCs , they are approximately 2.4 μm away—double the distance in PASMCs. This significant difference suggests a specialized arrangement in PASMCs that could influence cellular function.

2. Comparison of $I_{K_v}$$I_{K_v}$ Characteristics:

• Current Amplitude and Density: Patch-clamp recordings demonstrated that PASMCs have a significantly larger $I_{K_v}$$I_{K_v}$ amplitude compared to MASMCs. Even though MASMCs are larger in size ( as measured by membrane capacitance ) , the current density ( current per unit cell size ) was 8-18 times greater in PASMCs across various membrane potentials.

• Activation and Inactivation Parameters: The voltage dependence of $I_{K_v}$$I_{K_v}$ activation and inactivation differed between the cell types. In MASMCs , $I_{K_v}$$I_{K_v}$ activation occurred at more positive potentials , and inactivation occurred at more negative potentials compared to PASMCs. This indicates that Kv channels in PASMCs are more readily activated and less inactivated within the physiological voltage range.

3. Enhanced Effect of Mitochondrial Inhibitors on $I_{K_v}$$I_{K_v}$ in PASMCs:

• Antimycin A and Oligomycin A: These mitochondrial inhibitors had a significantly greater impact on $I_{K_v}$$I_{K_v}$ in PASMCs than in MASMCs. In PASMCs , the inhibitors caused notable shifts in the voltage dependence of $I_{K_v}$$I_{K_v}$ activation and inactivation and substantially reduced $I_{K_v}$$I_{K_v}$ amplitude. In MASMCs , these effects were present but markedly less pronounced.

• Functional Coupling: The stronger effect of mitochondrial inhibition on $I_{K_v}$$I_{K_v}$ in PASMCs suggests a tighter functional coupling between mitochondria and Kv channels in these cells.

4. Cytoskeletal Influence on Mitochondrial Positioning and $I_{K_v}$$I_{K_v}$ Modulation:

• Cytochalasin B Treatment: Disruption of the cytoskeleton with cytochalasin B in PASMCs led to a redistribution of mitochondria away from the plasma membrane. This change was associated with a significant attenuation of antimycin A's effect on $I_{K_v}$$I_{K_v}$ activation and inactivation parameters. However , the overall inhibition of $I_{K_v}$$I_{K_v}$ amplitude by antimycin A was not significantly affected by cytochalasin B.

• Implications: These findings imply that the cytoskeleton plays a role in maintaining the close proximity of mitochondria to the plasma membrane in PASMCs , which is necessary for the modulation of Kv channel function by mitochondrial signals.

5. Role of Intracellular Magnesium ( Mg²⁺ ) in $I_{K_v}$$I_{K_v}$ Regulation:

• Mg²⁺ Chelation Experiments: Dialysis of PASMCs with Mg²⁺ chelators ( EDTA or Na₂ATP ) reduced the effects of mitochondrial inhibitors on $I_{K_v}$$I_{K_v}$ inactivation , suggesting that Mg²⁺ is involved in this process.

• Mg²⁺ Supplementation: Increasing intracellular Mg²⁺ concentration by including higher MgCl₂ levels in the pipette solution mimicked the effects of mitochondrial inhibition on $I_{K_v}$$I_{K_v}$ inactivation. This supports the idea that mitochondrial activity influences intracellular Mg²⁺ levels , which in turn modulate Kv channel function.

Conclusion

This study concludes that the unique spatial distribution of mitochondria in PASMCs—specifically their close proximity to the plasma membrane—provides a structural basis for a stronger functional interaction between mitochondria and Kv channels in these cells compared to MASMCs. This close association allows mitochondrial signals , such as changes in Mg²⁺ concentration or reactive oxygen species ( ROS ) , to more effectively modulate Kv channel activity in PASMCs.

The functional coupling is evidenced by the greater impact of mitochondrial inhibitors on $I_{K_v}$$I_{K_v}$ in PASMCs. The attenuation of these effects by cytoskeletal disruption further indicates that the cytoskeleton is essential for maintaining mitochondrial positioning necessary for Kv channel regulation.

Intracellular Mg²⁺ emerges as a key mediator in the modulation of Kv channels by mitochondrial activity. The findings suggest that mitochondrial regulation of Mg²⁺ levels can influence the voltage-dependent activation and inactivation of Kv channels , thereby affecting cellular excitability.

The study highlights that the greater density of Kv channels and their tighter coupling with mitochondria in PASMCs may contribute to the pulmonary circulation's unique response to hypoxia , such as hypoxic pulmonary vasoconstriction. This mechanism contrasts with MASMCs , where the mitochondria are located further from the plasma membrane , Kv channel density is lower , and functional coupling with mitochondria is weaker , leading to different physiological responses to hypoxia.

In summary , the distinct structural and functional coupling between mitochondria and Kv channels in PASMCs is a crucial factor in the regulation of pulmonary artery excitability and may play a significant role in the unique oxygen-sensing mechanisms of the pulmonary circulation.

Antimycin A: An antibiotic that inhibits complex III of the mitochondrial electron transport chain, used to study mitochondrial function by blocking electron flow and inducing changes in cellular metabolism.

ATP (Adenosine Triphosphate): The primary energy carrier in cells, providing energy for various biochemical processes.

ATP Synthase: An enzyme complex in mitochondria that synthesizes ATP from ADP and inorganic phosphate during oxidative phosphorylation.

Brefeldin A BODIPY 558/568: A fluorescent dye conjugated to brefeldin A, used to stain the sarcoplasmic reticulum (SR) in cells for confocal microscopy imaging.

BAPTA-AM: A cell-permeable calcium chelator used to buffer intracellular calcium concentrations.

Ca²⁺ (Calcium Ion): A divalent cation that plays a crucial role in cellular signaling, muscle contraction, neurotransmission, and enzyme activity.

Confocal Microscopy: A fluorescence microscopy technique that increases optical resolution and contrast by using point illumination and a spatial pinhole to eliminate out-of-focus light, allowing for detailed three-dimensional imaging.

Cytochalasin B: A compound that disrupts actin filaments (F-actin), affecting the cytoskeleton and altering cellular processes such as shape, motility, and organelle distribution.

Cytoskeleton: A network of protein filaments within the cell, including microfilaments (actin), intermediate filaments, and microtubules, providing structural support, shape, and facilitating movement and intracellular transport.

Depolarization: A decrease in the membrane potential difference across the cell membrane, making the inside of the cell less negative relative to the outside, which can initiate action potentials in excitable cells.

Di-8-ANEPPS: A fluorescent voltage-sensitive dye that stains the plasma membrane, used in imaging studies to visualize cell membranes and assess membrane potential changes.

Electron Transport Chain (ETC): A series of protein complexes located in the inner mitochondrial membrane that transfer electrons through redox reactions, coupled with proton pumping to create an electrochemical gradient used to produce ATP.

Electrophysiology: The study of the electrical properties and activities of biological cells and tissues, often involving measurements of voltage changes or electric currents.

Endothelium: The thin layer of cells lining the interior surface of blood vessels, playing key roles in vascular tone regulation, blood flow, and barrier function.

Endothelium-Derived Contracting Factor (EDCF): A substance released by endothelial cells that causes vasoconstriction, counteracting the effects of vasodilators like nitric oxide.

F-actin Filaments: Filamentous actin structures that make up the microfilaments of the cytoskeleton, important for maintaining cell shape, enabling movement, and anchoring organelles.

Hyperpolarization: An increase in the membrane potential difference, making the inside of the cell more negative relative to the outside, which can inhibit action potential generation.

Hypoxia: A condition where tissues are deprived of an adequate oxygen supply, which can lead to various cellular and physiological responses.

Hypoxic Pulmonary Vasoconstriction (HPV): A physiological response where pulmonary arteries constrict in areas of low oxygen concentration, redirecting blood flow to better-oxygenated regions of the lung to optimize gas exchange.

IKv: Voltage-gated potassium current, representing the flow of potassium ions through voltage-gated potassium channels (Kv channels) in response to changes in membrane potential.

Inactivation Parameters: Characteristics that describe how an ion channel's activity decreases over time or under certain voltage conditions, including half-inactivation potential (Vh) and slope factor of inactivation (kh).

Intracellular Mg²⁺: Magnesium ions within the cell, essential for numerous enzymatic reactions, nucleic acid stability, and regulation of ion channels.

Juxtaposition: The close proximity or arrangement of two cellular components near each other, facilitating interaction or functional coupling.

Kv Channels (Voltage-Gated Potassium Channels): A family of transmembrane proteins that allow potassium ions to pass through the cell membrane in a voltage-dependent manner, crucial for setting the membrane potential and shaping action potentials.

L-type Ca²⁺ Channels: A type of voltage-dependent calcium channel found in the cell membrane, allowing calcium influx during depolarization, important for muscle contraction and signaling.

MASMCs (Mesenteric Arterial Smooth Muscle Cells): Smooth muscle cells isolated from mesenteric arteries, which supply blood to the intestines; used as a model for systemic circulation studies.

Membrane Depolarization: A reduction in the electrical potential across the cell membrane, leading to activation of voltage-sensitive channels and potential initiation of action potentials.

Membrane Potential: The electrical potential difference across a cell's plasma membrane due to the distribution of ions, fundamental to the function of excitable cells.

Mitochondrial Electron Transport Chain (mETC): The sequence of protein complexes in mitochondria that transfer electrons from electron donors to electron acceptors via redox reactions, coupled with proton translocation to generate ATP.

Mitochondrial Inhibitors: Substances that interfere with mitochondrial function by inhibiting components of the electron transport chain or ATP synthase, affecting ATP production and cellular metabolism.

MitoTracker Green FM: A fluorescent dye that selectively stains mitochondria in live cells, used for imaging mitochondrial distribution, morphology, and function.

mETC Inhibitors: Compounds that inhibit specific complexes within the mitochondrial electron transport chain, such as antimycin A (complex III inhibitor) and rotenone (complex I inhibitor).

NADH Oxidoreductase (Complex I): The first enzyme complex in the mitochondrial electron transport chain that oxidizes NADH, transferring electrons to ubiquinone.

NADPH Oxidase: An enzyme complex that produces reactive oxygen species (ROS) by transferring electrons from NADPH to molecular oxygen, involved in cellular signaling and host defense.

Oligomycin: An antibiotic that inhibits ATP synthase (complex V) in mitochondria, preventing ATP production, used to study mitochondrial function and bioenergetics.

PASMCs (Pulmonary Arterial Smooth Muscle Cells): Smooth muscle cells isolated from pulmonary arteries, which carry deoxygenated blood from the heart to the lungs; used to study pulmonary circulation and hypoxia responses.

Peripheral Mitochondria: Mitochondria located near the plasma membrane, as opposed to those in deeper cytoplasmic regions; may have specialized roles due to their proximity to the cell surface.

Pretone: The baseline or initial level of vascular tone (constriction) present in a blood vessel before any experimental manipulation or stimulus; represents the resting tension in the vessel wall that can influence responses to vasoconstrictors or vasodilators.

Reactive Oxygen Species (ROS): Chemically reactive molecules containing oxygen, such as superoxide anions and hydrogen peroxide, produced as byproducts of mitochondrial respiration and involved in cell signaling and oxidative stress.

Resting Membrane Potential: The membrane potential of a cell at rest, typically negative inside relative to outside, determined by ion concentration gradients and membrane permeability.

Sarcoplasmic Reticulum (SR): A specialized form of endoplasmic reticulum in muscle cells that stores and releases calcium ions to trigger muscle contraction.

Steady-State Activation: The relationship between the probability of an ion channel being open and the membrane potential under equilibrium conditions; characterized by parameters like half-activation potential (Va) and slope factor of activation (ka).

Superoxide: A reactive oxygen species (O₂⁻) generated by the one-electron reduction of molecular oxygen, can contribute to oxidative stress and signaling pathways.

Voltage Clamp: An experimental technique that allows control of the membrane potential of a cell while measuring the ionic currents flowing through its channels, used to study ion channel properties.

Voltage-Dependent Characteristics: Properties of ion channels that change in response to the membrane potential, including activation and inactivation kinetics and voltage sensitivity.

Voltage-Dependent Inactivation: The process by which an ion channel becomes less responsive or closes in response to sustained depolarization, reducing ion flow.

Vasoconstriction: The narrowing of blood vessels due to contraction of the muscular wall, reducing blood flow and increasing vascular resistance.

Vasodilation: The widening of blood vessels due to relaxation of the muscular wall, increasing blood flow and decreasing vascular resistance.

[Ca²⁺]i (Intracellular Calcium Concentration): The concentration of calcium ions within the cell cytoplasm, a key signaling molecule regulating numerous cellular processes, including muscle contraction and neurotransmitter release.

X-Y Optical Slices: Images taken in the horizontal plane (x and y axes) at different depths along the z-axis during confocal microscopy, allowing visualization of structures within a specific focal plane.

X-Z and Y-Z Optical Cross Sections: Images taken along vertical planes (x-z and y-z axes) to provide depth information and three-dimensional structure in microscopy.

Z-sectioning: A microscopy technique in confocal imaging where a series of optical slices are taken at different depths (along the z-axis) to create a three-dimensional reconstruction of a specimen, allowing detailed analysis of spatial relationships within cells.