Endogenous nitric oxide formation in cardiac myocytes does not control respiration during ß-adrenergic stimulation.

KEY POINTS: In the heart, endothelial nitric oxide (NO) controls oxygen consumption in the working heart through paracrine mechanisms. While cardiac myocytes contain several isoforms of NO synthases, it is unclear whether these can control respiration in an intracrine fashion. A long-standing controversy is whether a NOS exists within mitochondria. By combining fluorescence technologies with electrical field stimulation or the patch-clamp technique in beating cardiac myocytes, we identified a neuronal NO synthase (nNOS) as the most relevant source of intracellular NO during ß-adrenergic stimulation, while no evidence for a mitochondria-located NOS was obtained. The amounts of NO produced by non-mitochondrial nNOS were insufficient to regulate respiration during ß-adrenergic stimulation, arguing against intracrine control of respiration by NO within cardiac myocytes. ABSTRACT: Endothelial nitric oxide (NO) controls cardiac oxygen (O2 ) consumption in a paracrine way by slowing respiration at the mitochondrial electron transport chain. While NO synthases (NOSs) are also expressed in cardiac myocytes, it is unclear whether they control respiration in an intracrine way. Furthermore, the existence of a mitochondrial NOS is controversial. Here, by combining fluorescence imaging with electrical field stimulation, the patch-clamp method and knock-out technology, we determined the sources and consequences of intracellular NO formation during workload transitions in isolated murine and guinea pig cardiac myocytes and mitochondria. Using 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF) as a fluorescent NO-sensor that locates to the cytosol and mitochondria, we observed that NO increased by ∼12% within 3 min of ß-adrenergic stimulation in beating cardiac myocytes. This NO stems from neuronal NOS (nNOS), but not endothelial (eNOS). After patch clamp-mediated dialysis of cytosolic DAF, the remaining NO signals (mostly mitochondrial) were blocked by nNOS deletion, but not by inhibiting the mitochondrial Ca2+ uniporter with Ru360. While in isolated mitochondria exogenous NO inhibited respiration and reduced the NAD(P)H redox state, pyridine nucleotide redox states were unaffected by pharmacological or genetic disruption of endogenous nNOS or eNOS during workload transitions in cardiac myoctyes. We conclude that under physiological conditions, nNOS is the most relevant source for NO in cardiac myocytes, but this nNOS is not located in mitochondria and does not control respiration. Therefore, cardiac O2 consumption is controlled by endothelial NO in a paracrine, but not intracrine, fashion.

Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelle Ca(2+) crosstalk.

RATIONALE: Mitochondrial Ca(2+) uptake is essential for the bioenergetic feedback response through stimulation of Krebs cycle dehydrogenases. Close association of mitochondria to the sarcoplasmic reticulum (SR) may explain efficient mitochondrial Ca(2+) uptake despite low Ca(2+) affinity of the mitochondrial Ca(2+) uniporter. However, the existence of such mitochondrial Ca(2+) microdomains and their functional role are presently unresolved. Mitofusin (Mfn) 1 and 2 mediate mitochondrial outer membrane fusion, whereas Mfn2 but not Mfn1 tethers endoplasmic reticulum to mitochondria in noncardiac cells. OBJECTIVE: To elucidate roles for Mfn1 and 2 in SR-mitochondrial tethering, Ca(2+) signaling, and bioenergetic regulation in cardiac myocytes. METHODS AND RESULTS: Fruit fly heart tubes deficient of the Drosophila Mfn ortholog MARF had increased contraction-associated and caffeine-sensitive Ca(2+) release, suggesting a role for Mfn in SR Ca(2+) handling. Whereas cardiac-specific Mfn1 ablation had no effects on murine heart function or Ca(2+) cycling, Mfn2 deficiency decreased cardiomyocyte SR-mitochondrial contact length by 30% and reduced the content of SR-associated proteins in mitochondria-associated membranes. This was associated with decreased mitochondrial Ca(2+) uptake (despite unchanged mitochondrial membrane potential) but increased steady-state and caffeine-induced SR Ca(2+) release. Accordingly, Ca(2+)-induced stimulation of Krebs cycle dehydrogenases during ß-adrenergic stimulation was hampered in Mfn2-KO but not Mfn1-KO myocytes, evidenced by oxidation of the redox states of NAD(P)H/NAD(P)(+) and FADH(2)/FAD. CONCLUSIONS: Physical tethering of SR and mitochondria via Mfn2 is essential for normal interorganelle Ca(2+) signaling in the myocardium, consistent with a requirement for SR-mitochondrial Ca(2+) signaling through microdomains in the cardiomyocyte bioenergetic feedback response to physiological stress.