Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection

Abstract

Mitochondrial nitric oxide synthase (mtNOS), its cellular NOS isoform, and the effects of mitochondrially produced NO on bioenergetics have been controversial since mtNOS was first proposed in 1995. Here we functionally demonstrate the presence of a NOS in cardiac mitochondria. This was accomplished by direct porphyrinic microsensor measurement of Ca(2+)-dependent NO production in individual mitochondria isolated from wild-type mouse hearts. This NO production could be inhibited by NOS antagonists or protonophore collapse of the mitochondrial membrane potential. The similarity of mtNOS to the neuronal isoform was deduced by the absence of NO production in the mitochondria of knockout mice for the neuronal, but not the endothelial or inducible, isoforms. The effects of mitochondrially produced NO on bioenergetics were studied in intact cardiomyocytes isolated from dystrophin-deficient (mdx) mice. mdx cardiomyocytes are also deficient in cellular endothelial NOS, but overexpress mtNOS, which allowed us to study the mitochondrial enzyme in intact cells free of its cytosolic counterpart. In these cardiomyocytes, which produce NO beat-to-beat, inhibition of mtNOS increased myocyte shortening by approximately one-fourth. Beat-to-beat NO production and altered shortening by NOS inhibition were not observed in wild-type cells. A plausible mechanism for the reversible NO inhibition of contractility in these cells involves the reaction of NO with cytochrome c oxidase. This suggests a modulatory role for NO in oxidative phosphorylation and, in turn, myocardial contractility.

Figure 1

Photomicrograph of a mitochondrial preparation. This image (×1,200), which was acquired by using phase-contrast optics, shows a tightly coupled (respiratory control ratio = 10) mitochondrial preparation with a microsensor positioned next to one of the organelles. To-scale representations of an erythrocyte and a cardiomyocyte are superimposed on the image to show their relative sizes in comparison to the organelles, demonstrating the feasibility of measuring NO production from a single mitochondrion. Isolated cardiac mitochondria are ≈2–3 μm in length, somewhat larger than their counterparts in fixed cells (≈0.75–1.5 μm).

Figure 2

Electrochemical demonstration of NO production by isolated mitochondria. A porphyrinic microsensor was used to measure NO production by individual tightly coupled cardiac mitochondria. The organelles were in aerobic Ca²⁺-free mitochondrial solution without supplemental l-arginine or cofactors, under which conditions there was no detectable NO production. When exogenous Ca²⁺ (10 μM) was added to the bath, it evoked a rapid production of NO (28 ± 9 nM; n = 8; traces A and B) that was inhibited by the addition of the NOS antagonists, N^G-monomethyl-l-arginine (l-NMA, 100 μM; trace A) or 7-nitroindazole (50 μM; not shown). The Ca²⁺-dependent production of NO was also inhibited by pretreatment with ruthenium red (RR, 1 μM; trace C), a blocker of the electrogenic uniporter that is responsible for Ca²⁺ uptake by mitochondria.

Figure 3

Identification of the nNOS isoform in cardiac mitochondria. The identification of the isoform of cardiac mtNOS was deduced by using mitochondria isolated from the hearts of knockout mice for the neuronal (nNOS^−/−), inducible (iNOS^−/−), and endothelial (eNOS^−/−) isoforms. Only the mitochondria isolated from the hearts of nNOS^−/− mice failed to produce NO (n = 5).

Figure 4

Beat-to-beat NO production in an mdx cardiomyocyte. Norepinephrine (NE; 1 μM) evoked NO (726 ± 260 nM; n = 7; A Left) production in a quiescent wild-type cardiomyocyte. Pacing (voltage = 2 × threshold; duration = 10 ms; f = 2 Hz) the cell did not evoke detectable NO formation (A Right). In mdx cardiomyocytes, however, NE (1 μM) evoked a diminished NO response (126 ± 90 nM; n = 7; not shown) or none at all (n = 5; B Left). All paced mdx cells released NO (141 ± 94 nM; n = 12; B Right) beat-to-beat.

Figure 5

Demonstration of the effects of mitochondrially produced NO on contractility. Administration of NOS antagonists, l-NMA (100 μM; n = 7; A) or 7-nitroindazole (50 μM; n = 6; not shown) effectively inhibited electrically stimulated beat-to-beat NO production in mdx cardiomyocytes and NE (1 μM) evoked NO release in mdx and wild-type cells (n = 7; not shown). An alternative way to block Ca²⁺-dependent NO production was by inhibiting the electrogenic Ca²⁺ uniporter. This was rapidly and reversibly accomplished by collapsing the membrane potential with the protonophore uncouplers, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 100 nM; n = 5; B) or carbonyl cyanide m-chlorophenylhydrazone (CCCP, 100 nM; n = 4; not shown). Although contractility in mdx cardiomyocytes was approximately half that of normal cells, inhibition of NO production with l-NMA increased contractility by approximately one-fourth in mdx cells (Left; n = 8), but had no effect on normal cells.

Figure 6

Simultaneous measurement of Ca²⁺, NO, and NO in an mdx cardiomyocyte. A single electrical stimulus (voltage = 2 × threshold; duration = 10 ms) from a bipolar microelectrode (area = 50 μm²) evoked a rise in cytoplasmic Ca²⁺ (411 ± 57; nM; trace A) followed by the production of NO (149 ± 97 nM; trace B) 10–20 ms thereafter. The onset of NO production, in turn, was followed, in 25–50 ms, by an increase in intracellular NO (112 ± 31 nM; n = 6; B). Although the NO transient rapidly peaked and returned to baseline, the NO signal rose to a plateau and gradually declined (decline not shown). There was no detectable NO production in quiescent cells. Electrical stimulation evoked Ca²⁺ transients but did not evoke detectable NO production in wild-type cells (not shown).

Figure 7

Alternate reactions catalyzed by cytochrome c oxidase. (A) The normal (accepted) reaction cycle involving the four-electron reduction of dioxygen to water. (B) Proposed three-electron reduction of dioxygen and NO to nitrite ion and water, possibly via a peroxynitrite intermediate. For NO to be inhibitory, the rate-determining step in B must be slower than in A.