Research ArticleCARDIAC IMAGING

Responsive monitoring of mitochondrial redox states in heart muscle predicts impending cardiac arrest

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Science Translational Medicine  20 Sep 2017:
Vol. 9, Issue 408, eaan0117
DOI: 10.1126/scitranslmed.aan0117
  • Fig. 1. Spectra of the oxygenated/oxidized and deoxygenated/reduced forms of hemoglobin, myoglobin, and mitochondria and their structural implications.

    Raman scatter reflects vibrations, stretching, or bending of specific bonds within the porphyrin ring (A), a structure present in hemoglobin, myoglobin, and mitochondrial cytochromes. Shown here are enhancement factor-corrected spectra for mitochondria isolated from rodent myocardium (B), equine myoglobin (C), and purified rodent hemoglobin (D). In (B) to (D), blue lines represent deoxygenated/reduced species, and red lines represent oxygenated/oxidized species. The intensity of each spectrum is normalized to 1 arbitrary unit (AU) by correcting for the enhancement factor of each species. The identified peaks are in agreement with previously identified vibrational modes, as described by Spiro (54), Kitagawa et al. (55), Adar and Erecinska (56), and Argade et al. (43) for mitochondrial cytochromes and by Spiro and Strekas (44, 54) for myoglobin and hemoglobin.

  • Fig. 2. Spectral processing algorithm.

    (A) Coarse baseline estimation: The processed, averaged, spectrum y gathered by the device includes a potent fluorescent baseline. A coarse estimate of this baseline, Embedded Image is obtained by jointly estimating fifth-order polynomial coefficients and library spectra coefficients using a linear regression. The first iteration of the true RR spectrum is thus given by Embedded Image. (B) Iterative baseline refinement: A composite regression curve ŷ0, which most closely fits y0, is then calculated from a linear combination of library spectra weighted by regression coefficients. A slow-varying cubic spline fit to the residual res0 = y0ŷ0, denoted by Embedded Image, is added to the initial baseline to include any slow wobbles in the baseline that were not captured by the polynomial fit. (B, inset) Components of ŷ0 include oxyhemoglobin (HbO), deoxyhemoglobin (Hb), oxymyoglobin (MbO), deoxymyoglobin (Mb), and oxidized and reduced mitochondrial spectra. (C) The iterative baseline fitting procedure is repeated thrice to obtain the final estimate (ŷR). The residual (res) is calculated by subtracting the final regression curve (ŷR) from the baseline-adjusted spectrum (yR).

  • Fig. 3. 3RMR response to graded ischemia ex vivo.

    (A) As coronary flow rate (CFR) was incrementally decreased from baseline in a flow-controlled retrograde Langendorff perfusion experiment, myocardial tissue oxygen tension (tPO2, gray bars) decreased from a baseline of 97.3 ± 5.6 mmHg (mean ± SD) to 1.4 ± 1.3 mmHg [P < 0.0001, repeated-measures analysis of variance (ANOVA)]. Contemporaneously, the 3RMR increased from a baseline of 22 ± 3% during full perfusion to 54 ± 14% during complete ischemia (P < 0.001, repeated-measures ANOVA). Data are means, and error is SD. (B) Increases in 3RMR were nonlinearly (single-phase decay) associated with decreases in contractility (dP/dTmax). Data are means of each 10-min observation period. The line is a single-phase decay line with 95% confidence interval (CI) of regression line; P < 0.0001, Spearman correlation coefficient r = 0.88; r2 = 0.69. (C) Contractility (dP/dTmax) was linearly associated with tPO2. Data are means of each 10-min observation period. Line is a linear regression line with 95% CI of regression line; P < 0.0001, Pearson correlation coefficient r = −0.88; r2 = 0.71; n = 10 rodents.

  • Fig. 4. 3RMR response during in vivo occlusion of the IVC.

    (A) After complete IVC occlusion (IVCO), tissue oxygen tension (tPO2, gray line) decreased from a baseline of 25 to 10 mmHg within 2 min. The increase in 3RMR signal (red line) was delayed from this decrease in tPO2 by 2 to 3 min (integration time, 3 min). Mean arterial blood pressure (mABP, black line) decreased abruptly after IVCO as is typical for acute changes in myocardial preload. n = 1 representative sample. (B) After 10 min of IVCO (post), myocardial tPO2 decreased significantly (P = 0.0006) and 3RMR increased significantly (P = 0.0039, paired t test). (C) Acute IVCO caused an abrupt decrease in myocardial preload, which significantly decreased contractility (defined here as dP/dTmax). From this new (that is, after occlusion) baseline, 3RMR significantly increased (P < 0.0001, red bars) and was associated with a significant decrease in contractility (P = 0.0027, blue bars, linear trend repeated-measures ANOVA). (B and C) Data are means, and error is SEM. n = 8 rodents.

  • Fig. 5. Correlations between tPO2 and RRS-based 3RMR, oxyhemoglobin, and oxymyoglobin concentrations in rodents.

    (A) After baseline measurements on 100% oxygen, arterial hypoxemia was induced by ventilation with 5 to 8% oxygen. As tissue oxygen tension (tPO2) decreased, 3RMR increased from baseline levels of ~25 to >40%. Among 1859 data points (1-min medians), 3RMR was <40% at all but 12 points in which tPO2 was >10 mmHg, making a 3RMR < 40% clinically reassuring (specific diagnostic test). When tPO2 < 10 mmHg, 3RMR exceeded 40% in most data points. (B) RRS-based tissue oxyhemoglobin saturation (SHbO2) was related to tPO2 using hyperbolic fit. (C) Tissue myoglobin saturation (SMbO2) and tPO2 were also correlated using a hyperbolic fit. (A to C) Line is a nonlinear regression line in the form y = xn/(k + xn), as expected for oxygen binding to heme. (A) n = 2.8, r2 = 0.59; (B) n = 1, r2 = 0.62; (C) n = 1, r2 = 0.7.

  • Fig. 6. Raman-based oxygenation and hemodynamic data of two groups of rodents in vivo, separated by 3RMR at 10 min.

    (A) 3RMR values over time in animals in which 3RMR at 10 min exceeded 40% (red line, n = 12) and those in which it was at or below 40% at 10 min of hypoxia (black line, n = 19) were significantly different (P < 0.0001, repeated-measures ANOVA with Bonferroni correction). (B) In animals with higher 3RMR, myocardial tPO2 was significantly lower than in animals with a low 3RMR at 10 min (P = 0.001, repeated-measures ANOVA with Bonferroni correction). (C) Myocardial contractility (dP/dTmax) was similar between groups at 10 min (P = 0.065) but was significantly lower in the high 3RMR group by 30 min (P < 0.0001), suggesting the inability to increase contractility in response to hypoxemia. (A to C) Data are means, and error (shaded) is SEM. (D) Animals in the higher 3RMR group exhibited a significantly higher incidence of cardiac arrest within the 30-min observation period compared with those in the lower 3RMR group (P < 0.0001, log-rank test). (E) Sensitivity (solid black) and specificity (dotted black) for 3RMR values at 10 min, as predictive of cardiac arrest within 30 min, were jointly maximized at a threshold 3RMR value of 40%. (F) Receiver operating characteristics plot of 3RMR (solid red, AUC 0.98), tissue oxygen tension (dotted black, AUC 0.93), tissue oxyhemoglobin saturation (blue dot-dash, AUC 0.82), contractility (dotted green, AUC 0.51), and ejection fraction (orange dot-dash, AUC 0.39) measurements at 10 min as diagnostic tests predicting impending cardiac arrest in the following 20 min.

  • Fig. 7. 3RMR performance in swine undergoing aortic cross-clamping and subsequent reperfusion.

    (A) In swine, 3RMR was 16.8 ± 6.2% at baseline, increased to 54.7 ± 15.1% after 75 min of ischemic time, and then decreased to 11.7 ± 3.5% 3 min after reperfusion (P = 0.0015, repeated-measures ANOVA with Dunnett’s correction). (B) After myocardial reperfusion with cardiopulmonary bypass, 3RMR decreased to near-baseline levels within 3 min (n = 1, representative sample). (C) RRS-based tissue oxyhemoglobin saturation (SHbO2) decreased during ischemia, also returning to baseline levels during reperfusion (P = 0.005), as did oxymyoglobin saturation (SMbO2, P = 0.03; D). (A, C, and D) *P < 0.05 and **P < 0.01. All data are means, and error is SEM. n = 5 swine.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/408/eaan0117/DC1

    Materials and Methods

    Fig. S1. Schematic of the RRS system.

    Fig. S2. Sensitivity and specificity plots of 3RMR to detect severe tissue hypoxia.

    Fig. S3. Survival curves over time for rodents during the hypoxia experiment when risk-stratified at 10 min using tissue PO2, tissue oxyhemoglobin saturation, or tissue oxymyoglobin saturation.

    Fig. S4. Intracellular ATP and NADH concentrations after hypoxia experiment.

    Fig. S5. Oxyhemoglobin and oxymyoglobin saturations in rodents separated by the 3RMR value.

    Fig. S6. Raw RR spectra after baseline correction, the best-fit line of the explained spectrum, and the residual.

    Fig. S7. Correlation between oxyhemoglobin saturations based on RR spectral analysis and traditional co-oximetry.

    Fig. S8. Screenshot of the user interface.

    Fig. S9. Quantile-quantile plots of the residual from the in vivo experiments.

    Fig. S10. Measured spectra and residua from the in vivo experiments.

    Fig. S11. Simultaneous changes in a single 3RMR and oxymyoglobin concentration reading are independent of changes in the total mitochondria–to–total myoglobin ratio.

    Fig. S12. Distribution of bootstrap estimates from single–point-in-time measurements during baseline and severe hypoxia.

    Table S1. Spectroscopically active components of myocardium.

    Table S2. Baseline characteristics of rodents included in the hypoxia experiment.

    Table S3. Depth of myocardial tissue sampling within a clinically relevant range of hemoglobin concentration and oxyhemoglobin saturation.

    Table S4. Scale factors for hemoglobin, myoglobin, and mitochondria.

    Table S5. Confidence limits of SHbO2, SMbO2, and 3RMR from a single instantaneous measurement during baseline and hypoxic conditions in a single animal.

    Table S6. Individual subject-level data.

    References (5761)

  • Supplementary Material for:

    Responsive monitoring of mitochondrial redox states in heart muscle predicts impending cardiac arrest

    Dorothy A. Perry, Joshua W. Salvin, Padraic Romfh, Peili Chen, Kalyani Krishnamurthy, Lindsay M. Thomson, Brian D. Polizzotti, Francis X. McGowan, Daryoosh Vakhshoori,* John N. Kheir*

    *Corresponding author. Email: dvakhshoori{at}pendar.tech (D.V.); john.kheir{at}childrens.harvard.edu (J.N.K.)

    Published 20 September 2017, Sci. Transl. Med. 9, eaan0117 (2017)
    DOI: 10.1126/scitranslmed.aan0117

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Schematic of the RRS system.
    • Fig. S2. Sensitivity and specificity plots of 3RMR to detect severe tissue hypoxia.
    • Fig. S3. Survival curves over time for rodents during the hypoxia experiment when risk-stratified at 10 min using tissue PO2, tissue oxyhemoglobin saturation, or tissue oxymyoglobin saturation.
    • Fig. S4. Intracellular ATP and NADH concentrations after hypoxia experiment.
    • Fig. S5. Oxyhemoglobin and oxymyoglobin saturations in rodents separated by the 3RMR value.
    • Fig. S6. Raw RR spectra after baseline correction, the best-fit line of the explained spectrum, and the residual.
    • Fig. S7. Correlation between oxyhemoglobin saturations based on RR spectral analysis and traditional co-oximetry.
    • Fig. S8. Screenshot of the user interface.
    • Fig. S9. Quantile-quantile plots of the residual from the in vivo experiments.
    • Fig. S10. Measured spectra and residua from the in vivo experiments.
    • Fig. S11. Simultaneous changes in a single 3RMR and oxymyoglobin concentration reading are independent of changes in the total mitochondria–to–total myoglobin ratio.
    • Fig. S12. Distribution of bootstrap estimates from single–point-in-time measurements during baseline and severe hypoxia.
    • Table S1. Spectroscopically active components of myocardium.
    • Table S2. Baseline characteristics of rodents included in the hypoxia experiment.
    • Table S3. Depth of myocardial tissue sampling within a clinically relevant range of hemoglobin concentration and oxyhemoglobin saturation.
    • Table S4. Scale factors for hemoglobin, myoglobin, and mitochondria.
    • Table S5. Confidence limits of SHbO2, SMbO2, and 3RMR from a single instantaneous measurement during baseline and hypoxic conditions in a single animal.
    • References (5761)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S6 (Microsoft Excel format). Individual subject-level data.

    [Download Table S6]

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