Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2.

RATIONALE: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. OBJECTIVE: How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. METHODS AND RESULTS: Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. CONCLUSIONS: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.

Mitochondrial respiration and membrane potential after low-flow ischemia are not affected by ischemic preconditioning.

Mitochondrial function following prolonged ischemia and subsequent reperfusion is better preserved by ischemic preconditioning (IP). In the present study, we analyzed whether or not IP has an impact on mitochondrial function at the end of a sustained ischemic period. Göttinger minipigs were subjected to 90-min low-flow ischemia without (n=5) and with (n=5) a preconditioning cycle of 10-min ischemia and 15-min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall (AW) and the control posterior wall (PW) at the end of ischemia. Basal mitochondrial respiration was not different between AW and PW. The ADP-stimulated (state 3) respiration in AW mitochondria compared to PW mitochondria was equally decreased in non-preconditioned and preconditioned pigs. The uncoupled respiration as well as the membrane potential (rhodamine 123 fluorescence) were not significantly different between groups. However, the recovery of the membrane potential (Delta rhodamine 123 fluorescence/s) after the addition of ADP was delayed in mitochondria obtained from AW compared to PW, both in non-preconditioned and in preconditioned pig hearts. Neither the amount of marker proteins for complexes of the electron transport chain nor the level of reactive oxygen species were affected by ischemia without or with IP. State 3 respiration and recovery of membrane potential were impaired in pig mitochondria after 90 min of low-flow ischemia. IP did not improve mitochondrial function during ischemia. Therefore, the preservation of mitochondrial function by IP may occur during reperfusion rather than during the sustained ischemic period.

Mitochondrial dysfunction induced by a cytotoxic adenine dinucleotide produced by ADP-ribosyl cyclases from cADPR.

ADP-ribosyl cyclases were previously shown to produce three new adenine dinucleotides, P1,P2 diadenosine 5'-diphosphate (Ap2A) and two isomers thereof (P18 and P24), from cyclic ADP-ribose (cADPR) and adenine (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 14509-14514). The Ap2A isomer P24, containing an unusual C1'-N3 N-glycosidic bond, is shown here to affect mitochondrial function through (i) opening of the permeability transition pore complex (and consequent proton gradient dissipation) and (ii) inhibition of Complex I of the respiratory chain. Whereas proton gradient dissipation is dependent upon the extracellular Ca(2+) influx triggered by P24, the effect on oxygen consumption is Ca(2+) independent. The proton gradient dissipation induces apoptosis in HeLa cells and thus appears to be responsible for the already described potent cytotoxic effect of P24 on several human cell types. The other products of ADP-ribosyl cyclase activity, Ap2A and cADPR, antagonize P24-induced proton gradient dissipation and cytotoxicity, suggesting that the relative concentration of P24, cADPR, and Ap2A in cyclase-positive cells may affect the balance between cell life and death.

Formation of reactive oxygen species at increased contraction frequency in rat cardiomyocytes.

OBJECTIVE: Reactive oxygen species (ROS) play an ambivalent role in cardiomyocytes: low concentrations are involved in cellular signaling, while higher concentrations contribute to cellular injury. We studied ROS formation during increases in contraction frequency in isolated cardiomyocytes. METHODS: Rat ventricular cardiomyocytes were loaded with dichlorodihydrofluorescein and electrically stimulated (37 degrees C). ROS formation was assessed by the rate of oxidation-dependent fluorescence increase (OxR). Oxygen consumption (VO(2)) and NAD(P)H autofluorescence were measured in parallel experiments. RESULTS: Increases in contraction frequency were accompanied by an increase in VO(2) and a decrease in NAD(P)H fluorescence. OxR increased to 124+/-4%, 146+/-8%, 204+/-25% and 256+/-29% of OxR at baseline during 1, 2, 3 and 4 Hz stimulation, and subsequently returned to baseline values with 0.2 Hz. The OxR increase was dose-dependently inhibited by the antioxidant NAC (10 and 100 mM), but unaffected by the NO synthase inhibitor l-NAME (200 microM and 10 mM). The OxR increase was attenuated when myosin ATPase activity was inhibited by butanedione monoxime (BDM; 5 mM). CONCLUSION: Increased contraction frequency induces ROS formation in rat cardiomyocytes.

Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning.

OBJECTIVE: Connexin 43 (Cx43) is involved in infarct size reduction by ischemic preconditioning (IP); the underlying mechanism of protection, however, is unknown. Since mitochondria have been proposed to be involved in IP's protection, the present study analyzed whether Cx43 is localized at mitochondria of cardiomyocytes and whether such localization is affected by IP. METHODS AND RESULTS: Western blot analysis on mitochondrial preparations isolated from rat, mouse, pig, and human hearts showed the presence of Cx43. The preparations were not contaminated with markers for other cell compartments. The localization of Cx43 to mitochondria was also confirmed by FACS sorting (double staining with MitoTracker Red and Cx43) and immuno-electron and confocal microscopy. To study the role of Cx43 in IP, mitochondria were isolated from the ischemic anterior wall (AW) and the control posterior wall (PW) of pig myocardium at the end of 90 min low-flow ischemia without (n=13) or with (n=13) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion. With IP, the mitochondrial Cx43/adenine nucleotide transporter ratio was 3.4+/-0.7 fold greater in AW than in PW, whereas the ratio remained unchanged in non-preconditioned myocardium (1.1+/-0.2, p<0.05). The enhancement of the mitochondrial Cx43 protein level occurred rapidly, since an increase of mitochondrial Cx43 was already detected with two cycles of 5 min ischemia/reperfusion in isolated rat hearts to 262+/-63% of baseline. CONCLUSION: These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.

4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one induces mitochondrial dysfunction and apoptosis upon its intracellular oxidation.

We investigated the mechanism of cell death induced by a furoquinolinone derivative, namely, 4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ), in the dark. Mitochondrial depolarization was found to be a causative event in HOFQ-induced apoptosis that was blunted either by replacing the 4-hydroxymethyl group with a methyl one, or by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH). In vitro enzymatic assay demonstrated that HOFQ is a substrate of ADH. In isolated mitochondria HOFQ was without effect, whereas in the presence of ADH and NAD(+) it caused the opening of the permeability transition pore, indicating that HOFQ-oxidized products affect mitochondrial function directly. Finally, an analogue bearing the formyl group at the C-4 position mimicked all the effects exerted by HOFQ. In conclusion, these results suggest that the direct action on mitochondria of HOFQ-oxidized products are responsible for their cytotoxicity, which might be exacerbated, but hardly determined, by photodynamic action and/or binding to DNA.

Induction of the mitochondrial permeability transition by the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Sorting cause and consequence of mitochondrial dysfunction.

The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) alters DNA and stimulates the activity of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme involved in DNA repair. The consumption of cellular NAD(+) by PARP-1 is accompanied by ATP depletion, mitochondrial depolarization and release of proapoptotic proteins, but whether a causal relationship exists among these events remains an open question. Most of cellular NAD(+) is stored in the mitochondrial matrix and becomes available for cytosolic and nuclear processes only after its release through the permeability transition pore (PTP), a voltage-gated inner membrane channel. Here we have explored whether MNNG affects mitochondrial function upstream of PARP-1 activation. We show that MNNG has a dual effect on isolated mitochondria. At relatively low concentrations (up to 0.1 mM), it selectively sensitizes the PTP to opening, while at higher concentrations (above 0.5 mM) it inhibits carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)-stimulated respiration. MNNG caused PTP opening and activation of the mitochondrial proapoptotic pathway in intact HeLa cells, which resulted in cell death that could be prevented by the PTP inhibitor CsA. We conclude that a key event in MNNG-dependent cell death is induction of PTP opening that occurs independently of PARP-1 activation.

Mitochondria and reperfusion injury. The role of permeability transition.

The viability of the ischemic myocardium is jeopardized by alterations, such as ATP decrease and elevation in intracellular [Ca(2+)], that are related to derangements in mitochondrial function. Besides these established notions, the elucidation of the apoptotic cascade and the availability of novel methodologies for in situ studies prompted new interest in mitochondria. The characterization of mitochondrial channels provided a contribution that is particularly relevant to cardiovascular research. Here we focus on the role of the permeability transition pore by analyzing the methodological requirements for its characterization, the consequences of its opening and the possible relationships with other mitochondrial functions, especially with the K(ATP) channels.