Paradoxical arteriole constriction compromises cytosolic and mitochondrial oxygen delivery in the isolated saline-perfused heart

Abstract

The isolated saline-perfused heart is used extensively to study cardiac physiology. Previous isolated heart studies have demonstrated lower tissue oxygenation compared with in vivo hearts based on myoglobin oxygenation and the mitochondrial redox state. These data, consistent with small anoxic regions, suggest that the homeostatic balance between work and oxygen delivery is impaired. We hypothesized that these anoxic regions are caused by inadequate local perfusion due to a paradoxical arteriole constriction generated by a disrupted vasoregulatory network. We tested this hypothesis by applying two exogenous vasodilatory agents, adenosine and cromakalim, to relax vascular tone in an isolated, saline-perfused, working rabbit heart. Oxygenation was monitored using differential optical transmission spectroscopy and full spectral fitting. Increases in coronary flow over control with adenosine (27 ± 4 ml/min) or cromakalim (44 ± 4 ml/min) were associated with proportional spectral changes indicative of myoglobin oxygenation and cytochrome oxidase (COX) oxidation, consistent with a decrease in tissue anoxia. Quantitatively, adenosine decreased deoxymyoglobin optical density (OD) across the wall by 0.053 ± 0.008 OD, whereas the reduced form of COX was decreased by 0.039 ± 0.005 OD. Cromakalim was more potent, decreasing deoxymyoglobin and reducing the level of COX by 0.070 ± 0.019 OD and 0.062 ± 0.019 OD, respectively. These effects were not species specific, as Langendorff-perfused mouse hearts treated with adenosine demonstrated similar changes. These data are consistent with paradoxical arteriole constriction as a major source of regional anoxia during saline heart perfusion. We suggest that the vasoregulatory network is disrupted by the washout of interstitial vasoactive metabolites in vitro. NEW & NOTEWORTHY Regional tissue anoxia is a common finding in the ubiquitous saline-perfused heart but is not found in vivo. Noninvasive optical techniques confirmed the presence of regional anoxia under control conditions and demonstrated that anoxia is diminished using exogenous vasodilators. These data are consistent with active arteriole constriction, occurring despite regional anoxia, generated by a disrupted vasoregulatory network. Washout of interstitial vasoactive metabolites may contribute to the disruption of normal vasoregulatory processes in vitro.

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

“…These data support the well-established notion that myoglobin has a much lower affinity for oxygen than COX, even in the intact heart, as myoglobin oxygenation occurred well before changes in mitochondria redox state ( Figure 5). This high level of deoxygenated myoglobin under control conditions is consistent with prior studies suggesting that the isolated saline perfused heart may be partially hypoxic even under control conditions 9,19,20,21,27,36 , underscoring the importance of monitoring cardiac tissue oxygenation when using this important model in cardiac physiology.…”

“…The VWTOS data is analyzed using full spectral fitting routine based on a reference library of spectra of cardiac chromophores and the spectrum of the light source as previously described 20,22,27,35 with a simple linear least squares approach. This spectral fitting procedure compensated for overlapping absorbance spectrum and does not rely on "isobestic" wavelengths.…”

Ischemia-Reperfusion Injuries Assessment during Pancreas Preservation

“…There are also a few methods that are less commonly applied by which oxygen tension can be estimated indirectly. The Balaban laboratory, for example, developed a system to assess the redox status of endogenous mitochondrial chromophores in the isolated perfused heart [ 59 ]. By using colour deconvolution, it is possible to measure the redox status of cytochrome c oxidase in the electron transport chain, which, due to its function of reducing oxygen to form water, therefore corresponds with mitochondrial oxygen tension.…”

Monitoring mitochondrial calcium and metabolism in the beating MCU-KO heart

“…As described, the fiber optic provides the ability to alternate white light with the laser, allowing us to measure changes in the cytochromes and cytoplasmic myoglobin with the alternating white light illumination. We analyzed the spectra data as described previously (Bauer et al, 2019;Femnou et al, 2017;Giles et al, 2018). As shown in Figure 4A, cytochromes bH, bL, and c1 are all components of complex III, cytochrome a605 is a component of complex IV, and cytochrome c is a mobile carrier of electrons between complex III and IV.…”

“…Coupling this method with extrinsic fluorescent probes of the ionic milieu, or other properties of the cell, expands the information content of this system. With regard to the heart and heart mitochondria, recent transmission optical spectroscopy visible light studies (Chess et al, 2013;Femnou et al, 2017;Giles et al, 2018;Kuzmiak-Glancy et al, 2018) have demonstrated the detection of numerous aspects of oxidative phosphorylation, in addition to the monitoring of cytosolic oxygen via myoglobin.…”