2.1.

Materials

Bovine serum albumin (BSA), dimethylsulfoxide (DMSO), 2,4-dinitrophenol (DNP), ethylene glycol bis-( $\beta$ -aminoethyl ether) N,N,N’N’-tetraacetic acid (EGTA), DL-glutamic acid, K-gluconate, carbonyl cyanide 4-trifluoromethoxyphenyl hydrazone (FCCP), N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), Mg-ATP, sodium pyruvate, and succinic acid were purchased from Sigma (St. Louis, Missouri, U.S.A.). Rhodamine 800 (Molecular Probes, Eugene, Oregon, U.S.A.) was dissolved in DMSO $\left(3.3\mathrm{mM}\right)$ , aliquoted and stored at $-{20}^{\circ }\mathrm{C}$ until further use when diluted to the required concentration in aqueous buffer. Collagenase type 3 was purchased from Worthington Biochemical Corporation, Lakewood, New Jersey, U.S.A.

2.2.

Buffers

Mitochondrial incubation buffer A (in mM): 20 HEPES-Na (pH 7.2), $20\mathrm{KCl}$ , 100 K-gluconate, and 1 EGTA-Na. Hepatocyte incubation buffer B (in mM): 30 HEPES-Na (pH 7.4), $130\mathrm{NaCl}$ , $6.7\mathrm{KCl}$ , $0.7{\mathrm{KH}}_2\mathrm{PO}4$ , $0.7{\mathrm{Na}}_2{\mathrm{SO}}_4$ , $4.8{\mathrm{CaCl}}_2$ , $0.6{\mathrm{MgCl}}_2$ , and 5.5 glucose. Cardiomyocyte incubation buffer C (in mM): 25 HEPES-Na (pH 7.4), $118\mathrm{NaCl}$ , $4.7\mathrm{KCl}$ , $1.2{\mathrm{MgSO}}_4$ , $0.2{\mathrm{CaCl}}_2$ , 15 glucose, 2 carnitine, 5 taurine, 2 creatine, 15 butadione monooxime, and BSA $(10\mathrm{mg}∕\mathrm{ml})$ . Cardiomyocyte high-potassium buffer D (in mM): 20 HEPES-Na, $120\mathrm{KCl}$ , 1 EGTA, $1{\mathrm{MgSO}}_4$ , 11 glucose. Krebs-Henseleit buffer (KHB, for isolated heart perfusion) (in mM): $25{\mathrm{NaHCO}}_3$ , $118\mathrm{NaCl}$ , $4.7\mathrm{KCl}$ , $1.75{\mathrm{CaCl}}_2$ , $1.2{\mathrm{MgSO}}_4$ , 0.5 EDTA, and 11 glucose.

2.3.

Isolation of Rat Liver Mitochondria

Male Sprague-Dawley rats $(320–370\mathrm{g})$ were anesthetized with pentobarbital solution $(120\mathrm{mg}∕\mathrm{kg})$ intraperitoneally. The liver was taken immediately following the heart excision from the same animal. Liver mitochondria were isolated as described elsewhere.¹¹ Mitochondrial preparations were suspended in the medium containing 20-mM HEPES-Na (pH 7.2), $120\mathrm{mM}$ $\mathrm{KCl}$ and $1\mathrm{mM}$ Na-EGTA to bring the concentration to $30–50\mathrm{mg}$ of protein/ml and stored on ice. Mitochondrial protein was determined by bicinchoninic acid assay using Sigma kit BCA-1. Mitochondrial oxygen consumption was measured using a Clark-type oxygen electrode (Yellow Springs Instruments, Ohio, U.S.A.) at $30°\mathrm{C}$ in the mitochondrial incubation buffer A. Glutamate-Na $\left(5\mathrm{mM}\right)+5\mathrm{mM}$ malate-Na or $5\mathrm{mM}$ succinate-Na were used as oxidizable substrates. Following stabilization of state 2 respiration, $0.3\mathrm{mM}$ MgADP was added to stimulate respiration (state 3), which returned to the level close to state 2 (state 4) upon completion of ADP phosphorylation. FCCP $\left(1\mathrm{\mu }\mathrm{M}\right)$ was added at the end of the assay to obtain uncoupled respiration rate. The respiration rates were expressed in nmol ${\mathrm{O}}_2\times {\min }^{-1}\times {\mathrm{mg}}^{-1}$ and the respiratory control index (RCI) as a ratio of state 3 to state 2 respiration rate. RCI was 6.9 $\pm 1.11$ $(n=5)$ with glutamate $+$ malate and $4.52\pm 0.49$ $(n=4)$ with succinate.

2.4.

Isolation of Rat Hepatocytes and Cardiomyocytes

Hepatocytes were isolated according to a procedure described elsewhere,¹² based on a modified collagenase perfusion method.¹³ Isolated hepatocytes were resuspended in hepatocyte incubation buffer B. Cell viability of the hepatocyte preparations checked by the Evans Blue exclusion test was over 60%.

Cardiomyocytes were isolated by the collagenase perfusion method¹⁴ and resuspended in buffer C. Cell viability counts were performed using a hemocytometer and a light microscope. Approximately 70% of isolated cardiomyocytes were rod-shaped, and were therefore considered viable. All measurements were performed immediately after cell isolation.

2.5.

Measurements of Rhod800 Binding in Isolated Rat Liver Mitochondria, Hepatocytes, and Cardiomyocytes

Rhod800 binding in mitochondria was determined as follows: mitochondria $(0.2\mathrm{mg}∕\mathrm{ml})$ were incubated in $1\mathrm{ml}$ of aerated buffer A plus $1\mathrm{mM}$ ${\mathrm{MgSO}}_4$ with $5\mathrm{\mu }\mathrm{M}$ rhod800 for $5\min$ in the presence of $5\mathrm{mM}$ MgATP and oxidizable substrates( $5\mathrm{mM}$ $\mathrm{glutamate}\text{-}\mathrm{Na}+5\mathrm{mM}$ succinate-Na) at $30°\mathrm{C}$ . FCCP $\left(1\mathrm{\mu }\mathrm{M}\right)$ was added simultaneously with rhod800. The reaction mixture, in open scintillation vials (25-mm diameter), was continuously shaken to provide adequate aeration. Following 5-min incubation, the mixture was rapidly cooled and centrifuged for $5\min$ at $10,000\times \mathrm{g}$ . Rhod800 was extracted from the mitochondrial pellet with 95% ethanol $\left(1\mathrm{ml}\right)$ and its content was then determined from the absorbance at $685\mathrm{nm}$ in the suspension supernatant and mitochondrial extract (Beckman DU 650 spectrophotometer, Beckman Instruments Inc., Missisauga, Ontario, Canada) $(ϵ=99{\mathrm{mM}}^{-1}{\mathrm{cm}}^{-1})$ .

Isolated cells were incubated on a slowly shaking water bath at $35°\mathrm{C}$ . Hepatocytes were incubated in the hepatocyte incubation buffer B. To collapse sarcolemmal membrane potential, cardiomyocytes were diluted 1:11 in $1.2\mathrm{ml}$ of buffer D in the presence or absence of FCCP $\left(1\mathrm{\mu }\mathrm{M}\right)$ . Rhod800 was added to a concentration of $1\mathrm{\mu }\mathrm{M}$ and allowed to equilibrate for $5\min$ . Samples were then centrifuged for $5\min$ at $100\times \mathrm{g}$ . Rhod800 content in the supernatant was determined spectrophotometerically. Rhod800 bound to the cells was extracted with $1\mathrm{ml}$ of 95% ethanol. After $30\min$ , the cells were centrifuged, absorbance of the extract was measured at $680–685\mathrm{nm}$ and used to determine the amount of dye. Ethanol extraction of the dye was close to 100%.

To calculate the ratio of bound to free rhod800 (B/F) in cell preparations corrected for cell viability, $\mathrm{B}∕{\mathrm{F}}_{\mathrm{corr}}$ , we assumed that nonviable cells were uncoupled, then

2.6.

Measurements of Rhod800 Fluorescence in Rat Liver Mitochondria, Hepatocytes, and Cardiomyocytes

For all measurement, mitochondria were incubated in buffer A plus $5\mathrm{mM}$ ATP, $5\mathrm{mM}$ glutamate, and $5\mathrm{mM}$ succinate, hepatocytes in buffer B, and cardiomyocytes in buffer C plus $5\mathrm{mM}$ pyruvate or buffer D. Preparations were diluted 1:10 in a cuvette containing high-potassium buffers with a continuously stirring magnetic bar, to a final concentration of protein: mitochondria $0.03–0.09\mathrm{mg}∕\mathrm{ml}$ , hepatocytes and cardiomyocytes to $0.3–0.7\mathrm{mg}∕\mathrm{ml}$ . Fluorescence was measured by exciting the sample in the respective incubation buffer in a disposable plastic cuvette $(1\mathrm{cm}\times 1\mathrm{cm}\times 4\mathrm{cm})$ using a 670-nm laser diode ( $5\mathrm{mW}$ , NVG Inc., Hazlehurst, Georgia, U.S.A.); emitted fluorescence (at approximately $718\mathrm{nm}$ ) was detected at 90° and transmitted through a fiber optic cable to a spectrometer (Control Development, South Bend, Indiana, U.S.A). To reduce scattered laser light intensity, a 695-nm high-pass filter (CRG 695) was introduced before the detector. The fluorescence spectrum of $1\mathrm{\mu }\mathrm{M}$ rhod800 was taken before the addition of the sample. Additional dye was added to ensure the $1\text{-}\mathrm{\mu }\mathrm{M}$ rhod800 concentration was maintained after addition of the sample. Spectra were taken every $20\sec$ for $5\min$ . After $5\min$ , FCCP was added to a final concentration of $1\mathrm{\mu }\mathrm{M}$ to reduce $\mathrm{\Delta }\Psi$ to near zero. Measurements were taken after each chemical addition.

2.7.

Heart Perfusion

Rat hearts $(1.4–1.7\mathrm{g})$ were removed quickly and perfused in a Langendorff mode with KHB aerated with a mixture of 95% ${\mathrm{O}}_2$ and 5% ${\mathrm{CO}}_2$ at 36°C. Following the placement of a left ventricular apical drain, a latex balloon was inserted through the mitral valve into the left ventricular cavity and filled with ${\mathrm{H}}_2\mathrm{O}$ . The balloon was connected to a Statham P23Db pressure transducer and to a Digi-Med Model-210 heart performance analyser (Micro-Med, Louisville, Kentucky, U.S.A.) to monitor heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and perfusion pressure (PP). Pressure-rate-product (PRP), calculated as developed pressure (LVSP minus LVEDP) multiplied by HR, was used as an index of mechanical work. The coronary flow rate was monitored using an ultrasonic blood flow meter (Transonic Systems Inc., Ithaca, New York, U.S.A.), and PP was measured continuously through the catheter connecting the aortic line and the second pressure transducer.

2.8.

Myocardial Absorbance Measurements

Reflectance and absorbance spectra in perfused rat hearts, in the range $400–1000\mathrm{nm}$ , were acquired using a probe connected to a bifurcated fiber-optic cable. One end of the cable was connected to a source of white light (Fiber Optic Illuminator, model 77501, Oriel Instruments, Stratford, Connecticut, U.S.A.), while a second end terminated at the detector. The individual fibers of the cable were combined into a common probe tip that was in a direct contact with a left ventricle. Nongated spectra were acquired every $30\sec$ $\left(60\mathrm{scans}\right)$ .

2.9.

Measurements of Rhod800 Fluorescence and Apparent Absorbance at the Laser Wavelength in Intact Hearts

Fluorescence measurements were performed by exciting rhod800 in the hearts with a laser diode (690 or $670\mathrm{nm}$ , $5\mathrm{mW}$ , NVG Inc., Georgia, U.S.A.). Signal acquisition was performed in two settings: at 90° (orthogonal mode) and 360° (reflection mode), respective to the laser beam direction.

In the first setting, a 690-nm laser was in contact with the anterior heart wall and the signal was acquired at 90° to the laser beam, at the surface of the left ventricle. The collected signal consisted of scattered excitation and emitted fluorescence $({\lambda }_{\mathrm{em}}=715–730\mathrm{nm})$ light from the deeper layers of the hearts. Nongated spectra were acquired in the range $600–800\mathrm{nm}$ every $30\sec$ ; acquisition time was $0.15–1\sec$ $(30–200\mathrm{scans}\mathrm{per}\mathrm{spectrum})$ . Apparent absorbance by rhod800 loaded in the hearts was estimated from the changes in the laser signal registered by the detector over the time-course of dye loading, using the equation

In the second setting, a front face illumination-acquisition mode, fibers transmitting excitation light from a 670-nm laser and fibers receiving reflected and emitted $({\lambda }_{\mathrm{em}}=720\mathrm{nm})$ light were combined in a single probe tip that was in a direct contact with a left ventricle. To reduce reflected light intensity, a 695-nm high-pass filter was introduced in the receiving fibers before the detector. The acquired signal consisted of a greatly reduced reflected laser light and emitted fluorescence from the epicardial layer of the heart. Nongated spectra were acquired in the range $600–800\mathrm{nm}$ every $4\min$ ; acquisition time was $30\sec$ $\left(8\mathrm{scans}\mathrm{per}\mathrm{spectrum}\right)$ .

All spectral data were processed using Grams/32 Version 4.11 computer program (Galactic Industries Corp., U.S.A.).

2.J.

Statistics

ANOVA (single factor) was used for data comparison. Differences were considered statistically significant when $P<0.05$ . Data are presented as means $\pm \mathrm{S}.\mathrm{E}.\mathrm{M}.$

3.1.

Rhod800 Binding and Fluorescence in Isolated Rat Mitochondria and Cells

In mitochondrial suspensions, almost all rhod800 (greater than 99%) was taken up by the mitochondria (Table 1 ). This uptake was $\mathrm{\Delta }\Psi$ -dependent: the average ratio of bound-to-free rhod800 (B/F) was over 40 in the energized mitochondria. After addition of FCCP, almost all of the dye was released into the supernatant (Table 1). The amount of rhod800 bound specifically to the viable cells (containing energized mitochondria) was much lower: B/F decreased dramatically in isolated cells in comparison to the isolated mitochondria (Table 1). In cell preparations, B/F, corrected for cell viability, was approximately 2 (67% of the total dye bound) for the cardiomyocytes and 3 (75% bound) for the hepatocytes (Table 1).

Table 1

Effects of FCCP on rhod800 binding and fluorescence in liver mitochondria, cardiomyocytes, and hepatocytes.1

Fluorescence of rhod800 $\left(1\mathrm{\mu }\mathrm{M}\right)$ in the incubation buffer decreased after addition of mitochondria and increased after addition of FCCP [Fig. 1a ]. Similar fluorescence responses were observed in cell preparations; however, the magnitude of the changes was lower [Fig. 1b]. The fluorescence peak was normalized to the initial fluorescence and $\mathrm{\Delta }{I}_F∕I_{F0}$ calculated (Table 1). In the mitochondrial preparations, $\mathrm{\Delta }{I}_F∕I_{F0}$ was almost 80% in the energized vs. 13% in the de-energized mitochondria (Table 1). In the hepatocytes and cardiomyocytes, the differences between $\mathrm{\Delta }{I}_F∕I_{F0}$ in coupled and FCCP-treated (uncoupled) cells were in the 10% range (Table 1). Again, this corresponds to a smaller fraction of rhod800 released into the buffer from the FCCP-treated cells than from the FCCP-treated mitochondria.

Fig. 1

Fluorescence of rhod800 in mitochondria and cardiomyocyte suspension. (a) Spectra of $1\text{-}\mathrm{\mu }\mathrm{M}$ rhod800 fluorescence in the incubation buffer before (solid), after (dot) addition of mitochondrial suspension, and after addition of $1\text{-}\mathrm{\mu }\mathrm{M}$ FCCP (dash-dot). (b) Spectra of $1\text{-}\mathrm{\mu }\mathrm{M}$ rhod800 fluorescence in the incubation buffer before (solid), after (dot) addition of isolated cardiomyocytes, and after addition of $1\text{-}\mathrm{\mu }\mathrm{M}$ FCCP (dash-dot). A spike under $750\mathrm{nm}$ was an artifact. See details in Sec. 2.

De-energization of mitochondria by FCCP extruded the dye from the matrix into the supernatant, where fluorescence quenching is lower. However, nearly all rhod800 was bound to mitochondrial membranes and proteins. We estimated the fraction of free matrix dye as 0.15 and 0.75% of the total mitochondria dye (assuming matrix volume of $1.5\mathrm{\mu }\mathrm{l}∕\mathrm{mg}$ ) in uncoupled $(\mathrm{\Delta }\Psi =0\mathrm{mV})$ and coupled $(\mathrm{\Delta }\Psi =-160\mathrm{mV})$ mitochondria respectively. This implies a very high partition coefficient of rhod800 between water and hydrophobic milieu such as membranes, perhaps due to the high hydrophobicity of the dye. Indeed, we estimated that rhod800 partition coefficient was approximately 5-fold higher than that of TMRM (Kupriyanov, unpublished observation). This property of rhod800 may explain relatively small changes in the binding and fluorescence in response to uncoupling of cardiomyocytes and hepatocytes (see Table 1). The dye binding to the plasma membrane, endo/sarcoplasmic reticulum and polyanions such as DNA/RNA greatly increased the fraction of passively bound dye, which increased $\mathrm{\Delta }\Psi$ -independent binding and fluorescence thereby reducing relative magnitude of the $\mathrm{\Delta }\Psi$ -dependent response. Therefore quantitative $\mathrm{\Delta }\Psi$ estimation with rhod800 in cell suspensions is more difficult than in mitochondria.

3.2.

Changes in the Myocardial Absorbance Spectra Caused by Rhod800

Perfusion of isolated rat hearts with KHB containing rhod800 changed the myocardial absorbance spectra resulting in the appearance of a prominent peak at $690\mathrm{nm}$ and an additional smaller peak at $630\mathrm{nm}$ [Fig. 2a ]. The peaks were well separated from the peaks of endogenous chromophores in the visible range, such as oxy- and deoxymyoglobin, and cytochrome c oxidase. The dye concentration of $55\mathrm{nM}$ was selected to bring its free cytosol concentration, after 60-min perfusion, to approximately $1\mathrm{\mu }\mathrm{M}$ , which is expected at the resting membrane potential of $-80\mathrm{mV}$ . Changes in the myocardial absorbance at $690\mathrm{nm}$ were almost linear during 60-min perfusion with $55\mathrm{nM}$ rhod800: ${\mathrm{R}}^2=0.98$ [Fig. 2b]. Thirty-min washout resulted in only partial ( $15\pm 3\%$ , $n=3$ ) dye washout, as determined from the changes in myocardial absorbance at $690\mathrm{nm}$ .

Fig. 2

Myocardial absorbance of a rat heart perfused with rhod800. Front-face white light illumination and acquisition mode was employed. (a) Representative cardiac absorbance spectra before (dash) and after (solid) 60-min loading with $55\mathrm{nM}$ rhod800. (b) Kinetics of myocardial absorbance at $690\mathrm{nm}$ during rhod800 $\left(55\mathrm{nM}\right)$ loading and washout.

3.3.

Deep Tissue Fluorescence and Apparent Absorption Versus Surface Fluorescence of Rhod800 in Rat Hearts

To measure rhod800 fluorescence in rat hearts, a laser diode was used for the dye excitation. In the first setting, we did not use a cut-off filter and the signal collected at 90° to the direction of the laser beam consisted of two peaks: a scattered laser light peak (at $690\mathrm{nm}$ ), and a fluorescence peak [Fig. 3a ]. This setup allowed us to obtain spectral information from the deeper layers of the hearts. The position of the fluorescence peak shifted from 715 to $730\mathrm{nm}$ during loading [Fig. 3a]. The intensity of the scattered excitation light $\left(I_{\mathrm{ex}}\right)$ passed through the myocardium and registered orthogonally to the laser beam direction was used to calculate changes in the apparent absorbance at 690-nm (excitation wavelength) due to the rhod800 accumulation, according to Eq. 3. This parameter provided a relative measure of the dye concentration in the hearts. Actual dye content was determined by ethanol-extraction of rhod800 from the hearts at the end of the experiments (Table 2 ). At $55\mathrm{nM}$ rhod800 in perfusate, average tissue concentration reached $10\mathrm{nmol}∕\mathrm{g}$ (Table 2), which at $ϵ=99{\mathrm{mM}}^{-1}{\mathrm{cm}}^{-1}$ gives an effective path-length at $690\mathrm{nm}$ of $\sim 1.8\mathrm{cm}$ . In this setting, the intensity of the fluorescence peak reached a maximum at approximately $25\min$ after perfusion with $55\mathrm{nM}$ rhod800 and decreased on further loading [Fig. 3b]. However, apparent absorbance at 690-nm increased linearly during the same time [Fig. 3c], indicating that the observed decrease in the fluorescence signal was most probably a result of an inner filter effect.¹⁵ Because of the significant increase in apparent absorbance during rhod800 loading, we concluded that the observed inner filter effect was due to absorption of the excitation (primary effect) and emission (secondary effect) light by the accumulated rhod800.¹⁶ Washout of rhod800 resulted in a linear decrease in apparent absorbance by $15\pm 3\%$ $(n=6)$ accompanied by an increase in fluorescence, probably as a result of an increase in the intensity of excitation light inside the tissue and excitation volume. However, at a given absorbance, fluorescence was higher during washout than during loading [Fig. 3d]. This effect may have been caused by the dye re-distribution at the washout stage (release from the mitochondria?) and a change of quantum efficiency taking place in the mitochondria or cytosol of perfused rat hearts. It has been reported that cholesterol could affect self-quenching of the fluorescence of lipid-conjugated rhodamines.¹⁷ In the heart tissue, different types of membrane are present: cholesterol-containing (cytoplasmic) and cholesterol-free (mitochondrial), as well as different polyanions (DNA and RNA). Re-distribution of rhod800 between these structures may have resulted in the fluorescence increase.

Fig. 3

Detection of rhod800 deep tissue fluorescence from a rat heart. A 690-nm laser diode placed against a left ventricle anterior heart wall was used for the dye excitation. The signal was acquired at 90° to the laser beam at the surface of the left ventricle. (a) Fluorescence emitted from a representative intact rat heart before (solid), after 20-min (dot), 40-min (dash), and 60-min (dash-dot) infusion of $55\mathrm{nM}$ rhod800. (b) Time course of a fluorescence signal at $720\mathrm{nm}$ . (c) Time course of apparent absorbance at $690\mathrm{nm}$ . (d) Fluorescence signal intensity plotted against apparent absorbance at $690\mathrm{nm}$ .

To reduce the inner filter effect and dye quenching, the hearts were perfused with $27.5\mathrm{nM}$ rhod800, which eliminated the fluorescence decline phase (not shown). Apparent absorbance at $690\mathrm{nm}$ linearly increased to approximately half of that observed during loading with $55\mathrm{nM}$ rhod800 (Table 2); thus, further confirming that this parameter was proportional to the amount of dye in the myocardium. In agreement with the apparent absorbance data, amount of rhod800 extracted from the hearts perfused with $27.5\mathrm{nM}$ rhod800 was nearly half of that extracted from the hearts perfused with $55\mathrm{nM}$ rhod800 (Table 2).

In the second setting, to eliminate the inner filter effect, a fluorescence signal was collected from the surface of the heart at 360° to the direction of a 670-nm laser beam. Fluorescence intensity was relatively weak in comparison to the reflected laser light intensity; thus, to improve the signal-to-noise ratio for the fluorescence signal, increase acquisition time and avoid detector saturation with the laser signal, a 695-nm high-pass filter was introduced before the detector. Detected signal consisted mostly of a fluorescence peak at $718\mathrm{nm}$ and a greatly reduced reflected laser signal at $670\mathrm{nm}$ [Fig. 4a ]. Fluorescence increased nearly linearly during 60-min loading with $55\mathrm{nM}$ rhod800: ${\mathrm{R}}^2=0.99$ [Fig. 4b]. Dissipation of $\mathrm{\Delta }\Psi$ with DNP at the loading stage did not significantly affect the fluorescence kinetics: the fluorescence peak reached $16,532\pm 4,913$ $(n=3)$ counts in the hearts treated with DNP $\left(50\mathrm{\mu }\mathrm{M}\right)$ and $15,768\pm 893$ ( $n=7$ , N.S.) in the absence of DNP. After the loading stopped, the fluorescence reached the plateau [Fig. 4b]. Treatment of the hearts with DNP at the washout stage resulted in a small (approximately 5%), but statistically significant increase in the heart’s fluorescence [Fig. 4c]. In comparison to the isolated cells, the scarcely detectable response to DNP uncoupling in the intact hearts was probably due to further increased background binding of the dye to the vascular and extracellular matrix elements. To test the effect of dissipation of the sarcolemmal membrane potential, the hearts were perfused with a high potassium buffer $\left(\right[{\mathrm{K}}^{+}]=24.7\mathrm{mM})$ , which resulted in the sarcolemmal membrane depolarization by approximately $43\mathrm{mV}$ and caused cardiac arrest. No changes were observed in the heart’s fluorescence during high potassium perfusion [Fig. 4c].

Fig. 4

Detection of rhod800 surface fluorescence from a rat heart. Fibers transmitting excitation light from a 670-nm laser and fibers receiving reflected and emitted light were combined in a single probe tip that was in a direct contact with a left ventricle. (a) Fluorescence emitted from a representative intact rat heart before (solid), after 30 (dot), and 60-min (dash) of infusion of $55\mathrm{nM}$ rhod800. (b) Average time course of a fluorescence signal at $720\mathrm{nm}$ $(n=7)$ . (c) Average apparent absorbance at $690\mathrm{nm}$ of the hearts treated with DNP ( $50\mathrm{\mu }\mathrm{M}$ final concentration, $n=4$ ) or perfused with high-potassium KHB ( $\left[{\mathrm{K}}^{+}\right]=24.7\mathrm{mM}$ , $n=3$ ) at the indicated periods of time.

Table 2

Effect of the dye loading conditions on rhod800 accumulation in rat hearts.1

3.4.

Cardiac Function

Following an approximately 10-min stabilization period, the hearts were perfused at a constant flow of $13–15\mathrm{ml}∕\min$ to provide the desired concentration of the dye/drugs during infusion. Baseline parameters were: $\mathrm{HR}=257\pm 13\mathrm{beats}\mathrm{per}\min$ (no pacing); $\mathrm{LVSP}=108\pm 6$ , $\mathrm{LVEDP}=6\pm 1$ , and mean $\mathrm{PP}=69\pm 1.3\mathrm{mmHg}$ $(n=8)$ . These parameters did not change significantly during perfusion with KHB for 1 hour. Sixty-min infusion of $55\mathrm{nM}$ rhod800 resulted in a moderate increase in LVEDP to $23\pm 3\mathrm{mmHg}$ , LVSP to $136\pm 9$ , and PP to $90\pm 5\mathrm{mmHg}$ $(n=5)$ . The HR declined to approximately 230 beats per minute, however, the change in HR was not significant. Infusion of $50\mathrm{\mu }\mathrm{M}$ DNP simultaneously with $55\mathrm{nM}$ rhod800 resulted in a decline in pressure-rate-product to $25\pm 14\%$ of the baseline $(n=3)$ due to a well-known uncoupling effect of DNP on the oxidative phosphorylation.