Cardioprotective mechanisms of mitochondria-targeted S-nitrosating agent and adenosine triphosphate-sensitive potassium channel opener are mutually exclusive

Figures

• 

  Individually, but not in combination, diazoxide and mitochondrial S-nitrosating agent improve cardiac function following ischemia.

• 

  Figure 1. Isolated myocyte and Langendorff isolated heart experimental protocols. A, Isolated mouse myocytes were exposed to 37°C control Tyrode's physiologic solution for 20 minutes (baseline), followed by a stress period (at 20 minutes; red arrow) with test solution (hypothermic hyperkalemic cardioplegia [CPG] with or without the adenosine triphosphate–sensitive potassium [KATP] channel opener diazoxide [DZX]) for 20 minutes, followed by a 20-minute reexposure to Tyrode's solution. Mitochondria-targeted S-nitrosating agent (MitoSNO) was infused at the end of the stress period (at 40 minutes; red arrow). Volume measurements were obtained every 5 minutes (bluesymbols). Contractility measurements were obtained at 5 minutes (baseline), 10 minutes, and 20 minutes after reexposure to Tyrode's solution (time, 50 and 60 minutes; green stars). B, Isolated mouse hearts underwent Langendorff baseline perfusion with Krebs–Henseleit buffer (KHB) solution for 30 minutes, followed by arrest (at 30 minutes; red arrow) with test solution (hypothermic hyperkalemic CPG with or without KATP channel opener DZX) and 90 minutes of global ischemia, followed by infusion of MitoSNO 1 μM or KHB (at 120 minutes;…

• 

  Figure 2. Hyperkalemic cardioplegia (CPG) exposure results in reduced myocyte contractility that is prevented by the adenosine triphosphate–sensitive potassium (KATP) channel opener diazoxide (DZX), and this protection is lost with the addition of mitochondrial-targeted S-nitrosating agent (MitoSNO). Myocytes were perfused with control 37°C Tyrode's solution (time 0-20 minutes), test solution (Tyrode's, CPG, or CPG + DZX; n = 5-20 each group; time 20-40 minutes; stress), followed by administration of MitoSNO (1 μM) or saline (at 40 minutes) and then 37°C Tyrode's solution (time 40-60 minutes). Contractility (velocity of shortening, percent shortening, or velocity of relengthening) was measured after 50 minutes (A) and 60 minutes (B) of reperfusion with Tyrode's solution. The middle horizontal line in the box represents the median ± SD percent change from baseline. The upper and lower whiskers represent the maximum and minimum values of nonoutliers, and each data point is represented as a dot. ∗Adjusted P < .05.

• 

  Figure 3. Hyperkalemic cardioplegia (CPG) with mitochondrial-targeted S-nitrosating agent (MitoSNO) results in reduced myocyte contractility compared with Tyrode's solution + MitoSNO that is worsened by the addition of adenosine triphosphate–sensitive potassium (KATP) channel opener diazoxide (DZX). Myocytes were perfused with control 37°C Tyrode's solution (time 0-20 minutes), test solution (Tyrode's, CPG, or CPG + DZX; n = 5-20 each group; time 20-40 minutes; stress), followed by administration of MitoSNO (1 μM) or saline (at 40 minutes) and then 37°C Tyrode's solution (time 40-60 minutes). Contractility (velocity of shortening, percent shortening, or velocity of relengthening) was measured after 50 minutes (A) and 60 minutes (B) of reperfusion with Tyrode's. The middle horizontal line in the box represents the median ± SD percent change from baseline. The upper and lower whiskers represent the maximum and minimum values of nonoutliers, and each data point is represented as a dot. ∗Adjusted P < .05; ∗∗adjusted P < .01; ∗∗∗adjusted P < .001.

• 

  Figure 4. Both the adenosine triphosphate–sensitive potassium (KATP) channel opener diazoxide (DZX) and mitochondrial-targeted S-nitrosating agent (MitoSNO) are associated with preserved left ventricular (LV) end-diastolic pressure (EDP) following prolonged global ischemia that is lost with their combination. Isolated mouse hearts underwent Langendorff perfusion for 30 minutes, followed by arrest with test solution (cardioplegia [CPG] or CPG + DZX) and 90 minutes global ischemia, infusion of MitoSNO 1 μM or Krebs–Henseleit buffer, and then reperfusion for 30 minutes. LV EDP was recorded during baseline and following ischemia at identical balloon volumes; n = 8-10 for each. A, Change in LV EDP from baseline across all balloon volumes following global ischemia; fitted values (solid lines) of the linear mixed model with random intercept superimposed on a smoother (dotted lines) for each group. As balloon volumes increase, the average change of EDP in the CPG + DZX + MitoSNO group is separated from that in the other treatment groups. ∗Adjusted P < .05, CPG + DZX + MitoSNO vs all other groups at each balloon volume. B, Change in LV EDP from baseline across all balloon volumes following global…

• 

  Figure 5. Cardioprotective mechanisms of mitochondria-targeted S-nitrosating agent (MitoSNO) and adenosine triphosphate–sensitive potassium channel opener diazoxide (DZX) are mutually exclusive. Isolated myocytes (n = 5-20 each) underwent exposure to stress with or without DZX and with or without MitoSNO. Isolated mouse hearts (n = 8-10 each) underwent 90 minutes of global ischemia with or without DZX in cardioplegia (CPG) with or without MitoSNO. DZX and MitoSNO were each individually cardioprotective; however, the combination of the two was detrimental. These data inform the molecular mechanism of action of DZX to support future clinical trials.

• 

  Figure 6. Schema of the mitochondrial electron transport chain with the adenosine triphosphate–sensitive potassium (KATP) channel and actions of the KATP channel opener diazoxide (DZX) and mitochondria-targeted S-nitrosating agent (MitoSNO). An electron microscopy photograph of an isolated mitochondria from our laboratory shows that the figure is a simplified image of the mitochondrial electron transport chain and a proposed mitochondrial KATP channel on the inner mitochondrial membrane. DZX inhibits succinate dehydrogenase (SDH) and was administered during stress in both models in the current study. MitoSNO stalls complex I and prevents succinate accumulation and was administered at the onset of reperfusion in both models. DZX may provide cardioprotection via a mitochondrial KATP channel, via SDH (complex II) inhibition, or via another mechanism during ischemia. MitoSNO provides cardioprotection via reversible inhibition of complex I and prevention of SDH accumulation during the initial 30 minutes of reperfusion. Each is cardioprotective; however, together they have a synergistic negative effect. NADH, Nicotinamide adenine dinucleotide, reduced form; NAD+, nicotinamide adenine…

• 

  Figure E1. The addition of mitochondrial-targeted S-nitrosating agent (MitoSNO) to Tyrode's physiologic solution reduces myocyte volume. Myocytes were perfused with control 37°C Tyrode's solution (time 0-20 minutes) or test solution (Tyrode's), followed by administration of MitoSNO (1 μM or 10 μM) or saline (at 40 minutes; red arrow) and then reperfusion with 37°C Tyrode's solution (time 40-60 minutes). The mean ± SEM percent change in myocyte volume from baseline is shown. ∗Adjusted P < .05 for Tyrode's vs Tyrode's + MitoSNO 1 μM; ∗∗adjusted P < .05 for Tyrode's vs Tyrode's + MitoSNO 10 μM. SEM, Standard error of the mean.

• 

  Figure E2. The addition of mitochondrial-targeted S-nitrosating agent (MitoSNO) following stress of hypothermic, hyperkalemic cardioplegia (CPG) reduces myocyte volume. Myocytes were perfused with control 37°C Tyrode's solution (time 0-20 minutes), then test solution (CPG) (time 20-40 minutes; stress), followed by administration of MitoSNO (1 μM or 10 μM) or saline (red arrow, at 40 minutes) and then reperfusion with 37°C Tyrode's solution (time 40-60 minutes). The mean ± SEM percent change in myocyte volume from baseline is shown. ∗Adjusted P < .05 for CPG vs CPG + MitoSNO 1 μM; ∗∗adjusted P < .05 for CPG vs CPG + MitoSNO 10 μM. SEM, Standard error of the mean.

• 

  Figure E3. The addition of mitochondrial-targeted S-nitrosating agent (MitoSNO) following stress of hypothermic, hyperkalemic cardioplegia (CPG) with diazoxide (DZX) does not alter myocyte volume. Myocytes were perfused with control 37°C Tyrode's solution (time 0-20 minutes), test solution (CPG + DZX) (time 20-40 minutes; stress), followed by administration of MitoSNO (1 μM or 10 μM) or saline (at 40 min; red arrow), and then reperfusion with 37°C Tyrode's solution (time 40-60 minutes). The mean ± SEM percent change in myocyte volume from baseline is shown. SEM, Standard error of the mean.

• 

  Figure E4. Mitochondrial-targeted S-nitrosating agent (MitoSNO) administered following exposure to Tyrode's physiologic solution or hyperkalemic cardioplegia (CPG) improves myocyte contractility. Myocytes were perfused with control 37°C Tyrode's solution (time 0-20 minutes) and test solution (Tyrode's or CPG) (time 20-40 minutes), followed by administration of MitoSNO (1 μM or 10 μM) or saline (at 40 minutes), then reperfusion with 37°C Tyrode's solution (at 40-60 minutes) (n = 5-20 each). Contractility (velocity of shortening, percent shortening, or velocity of relengthening) was measured after 50 minutes (A and C) and 60 minutes (B and D) of reperfusion with Tyrode's solution. Values are reported as mean ± SEM percent change from baseline. ∗Adjusted P < .05; ∗∗adjusted P < .01. SEM, Standard error of the mean.

• 

  Figure E5. Coronary flow following global ischemia was similar between groups. Isolated mouse hearts underwent Langendorff perfusion for 30 minutes, followed by arrest with test solution (cardioplegia [CPG] or CPG + diazoxide [DZX]) and 90 minutes of global ischemia, then infusion of mitochondrial-targeted S-nitrosating agent (MitoSNO) 1 μM or Krebs–Henseleit buffer, then reperfusion for 30 minutes. Coronary flow was measured by an in-line coronary flow probe and is represented as a percentage of baseline flow for each minute after reperfusion. Here confidence intervals are removed for clarity.