The 5′-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a natural energy sensor in mammalian cells that plays a key role in cellular and systemic energy homeostasis. At the cellular level, AMPK supports numerous processes required for energy and redox homeostasis, including mitochondrial biogenesis, autophagy, and glucose and lipid metabolism. Thus, understanding the pathways regulating AMPK activity is crucial for developing strategies to treat metabolic disorders. Mounting evidence suggests the presence of a link between cyclic AMP (cAMP) and AMPK signaling. cAMP signaling is known to be activated in circumstances of physiological and metabolic stress due to the release of stress hormones, such as adrenaline and glucagon, which is followed by activation of membrane-bound adenylyl cyclase and elevation of cellular cAMP. Because the majority of physiological stresses are associated with elevated energy consumption, it is not surprising that activation of cAMP signaling may promote AMPK activity. Aside from the physiological role of the cAMP/AMPK axis, numerous reports have suggested its role in several pathologies, including inflammation, ischemia, diabetes, obesity, and aging. Furthermore, novel reports have provided more mechanistic insight into the regulation of the cAMP/AMPK axis. In particular, the role of distinct cAMP microdomains generated by soluble adenylyl cyclase in regulating basal and induced AMPK activity has recently been demonstrated. In the present review, we discuss current advances in the understanding of the regulation of the cAMP/AMPK axis and its role in cellular homeostasis and explore some translational aspects.

Aging is one of the major non-reversible risk factors for several chronic diseases, including cancer, type 2 diabetes, dementia, and cardiovascular diseases (CVD), and it is a key cause of multimorbidity, disability, and frailty (decreased physical activity, fatigue, and weight loss). The underlying cellular mechanisms are complex and consist of multifactorial processes, such as telomere shortening, chronic low-grade inflammation, oxidative stress, mitochondrial dysfunction, accumulation of senescent cells, and reduced autophagy. In this review, we focused on the molecular mechanisms and translational aspects of cardiovascular aging-related inflammation, i.e., inflammaging.

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.

Uncontrolled release of Ca²⁺ from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ_m. Fura-2 was used to monitor cytosolic [Ca²⁺]_i or mitochondrial calcium [Ca²⁺]_m, after quenching the cytosolic compartment with MnCl₂. Mitochondrial ROS [ROS]_m production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg²⁺ concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ_m collapse and disturbance of ATP recovery. Simultaneously, Ca²⁺ oscillations occurred, [Ca²⁺]_m and [ROS]_m increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca²⁺ cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca²⁺ uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca²⁺ cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS]_m. In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca²⁺ oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca²⁺ cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Apoptosis of cardiomyocytes significantly contributes to the development of post-ischaemic cardiomyopathy. Although mitochondria have been suggested to play a crucial role in this process, the precise mechanisms controlling the mitochondria-dependent apoptosis in cardiomyocytes under ischaemia/reperfusion are still poorly understood. Here we aimed to analyse the role of the soluble adenylyl cyclase (sAC). Adult rat cardiomyocytes were subjected to simulated in vitro ischaemia (SI) consisting of glucose-free anoxia at pH 6.4. Apoptosis was detected by DNA laddering, chromatin condensation, and caspases cleavage. SI led to the translocation of sAC to the mitochondria and mitochondrial depolarization followed by cytochrome c release, caspase-9/-3 cleavage and apoptosis during simulated reperfusion (SR). Pharmacological inhibition of sAC during SI, but not during SR, significantly reduced the SI/SR-induced mitochondrial injury and apoptosis. Similarly, sAC knock-down mediated by an adenovirus coding for shRNA targeting sAC prevented the activation of the mitochondrial pathway of apoptosis. Analysis of the link between sAC and apoptosis revealed a sAC and protein kinase A-dependent Bax phosphorylation at Thr(167) and its translocation to mitochondria during SI, which subsequently caused mitochondrial oxygen radical formation followed by cytochrome c release and caspase-9 cleavage during SR. These results suggest a key role of sAC in SI-induced mitochondrial Bax translocation and activation of the mitochondrial pathway of apoptosis in adult cardiomyocytes.

Apoptosis of vascular smooth muscle cells (VSMC) in advanced atherosclerotic plaques is an important cause of plaque instability. Oxysterols have been suggested as important inducers of apoptosis in VSMC, but the precise mechanism is still poorly understood. Here we aimed to analyse the role of the soluble adenylyl cyclase (sAC). VSMC derived from rat aorta were treated with either 25-hydroxycholesterol or 7-ketocholesterol for 24 h. Apoptosis was detected by TUNEL staining and caspases cleavage. Oxysterols treatment led to the activation of the mitochondrial pathway of apoptosis (cytochrome c release and caspase-9 cleavage) and mitochondrial ROS formation, which were suppressed by the pharmacological inhibition or knockdown of sAC. Scavenging ROS with N-acetyl-l-cysteine prevented oxysterol-induced apoptosis. Analyses of the downstream pathway suggest that protein kinase A (PKA)-dependent phosphorylation and the mitochondrial translocation of the pro-apoptotic protein Bax is a key link between sAC and oxysterol-induced ROS formation and apoptosis. To distinguish between intra-mitochondrial and extra-mitochondrial/cytosolic sAC pools, sAC was overexpressed in mitochondria or in the cytosol. sAC expression in the cytosol, but not in mitochondria, significantly promoted apoptosis and ROS formation during oxysterol treatment. These results suggest that the sAC/PKA axis plays a key role in the oxysterol-induced apoptosis of VSMC by controlling mitochondrial Bax translocation and ROS formation and that cytosolic sAC, rather than the mitochondrial pool, is involved in the apoptotic mechanism.