In vitro studies suggested that sub-millisecond pulses of radiation elicit less genomic instability than continuous, protracted irradiation at the same total dose. To determine the potential of ultrahigh dose-rate irradiation in radiotherapy, we investigated lung fibrogenesis in C57BL/6J mice exposed either to short pulses (≤500 ms) of radiation delivered at ultrahigh dose rate (≥40 Gy/s, FLASH) or to conventional dose-rate irradiation (≤0.03 Gy/s, CONV) in single doses. The growth of human HBCx-12A and HEp-2 tumor xenografts in nude mice and syngeneic TC-1 Luc⁺ orthotopic lung tumors in C57BL/6J mice was monitored under similar radiation conditions. CONV (15 Gy) triggered lung fibrosis associated with activation of the TGF-β (transforming growth factor–β) cascade, whereas no complications developed after doses of FLASH below 20 Gy for more than 36 weeks after irradiation. FLASH irradiation also spared normal smooth muscle and epithelial cells from acute radiation-induced apoptosis, which could be reinduced by administration of systemic TNF-α (tumor necrosis factor–α) before irradiation. In contrast, FLASH was as efficient as CONV in the repression of tumor growth. Together, these results suggest that FLASH radiotherapy might allow complete eradication of lung tumors and reduce the occurrence and severity of early and late complications affecting normal tissue.

Background— An emerging concept is that a neuronal isoform of nitric oxide synthase (NOS1) may regulate myocardial contractility. However, a role for NOS1-derived nitric oxide (NO) in heart failure (HF) has not been defined. Methods and Results— Using a model of myocardial infarction-induced HF, we demonstrated that cardiac NOS1 expression and activity increased in HF rats ( P <0.05 and P <0.001 versus shams, respectively). This was associated with translocation of NOS1 from the ryanodine receptor to the sarcolemma through interactions with caveolin-3 in HF hearts. With ex vivo and in vivo pressure-volume analysis, cardiac NOS1-derived NO was found to be negatively inotropic in shams but not HF hearts. Ventricular elastance (E _es ) was significantly reduced in HF rats ( P <0.05), and τ, the time constant of left ventricular relaxation, was prolonged (both P <0.05). Acute NOS1 inhibition significantly increased E _es by 33±3% and τ by 17±2% ( P <0.05) in shams, although these effects were significantly attenuated in HF hearts. β-Adrenergic stimulation induced a marked increase in systolic performance in sham hearts, with the responses being significantly blunted in HF hearts. E _es increased by 163±42% ( P <0.01) in sham hearts and 56±9% in HF hearts, and LV +dP/dt increased by 97±9% ( P <0.01) in shams and 37±7% ( P <0.05) in the HF group. Interestingly, preferential NOS1 inhibition enhanced the blunted responses of LV +dP/dt and E _es to β-adrenergic stimulation in HF rats but had no effect in shams. Conclusions— These results provide the first evidence that increased NOS1-derived NO production may play a role in the autocrine regulation of myocardial contractility in HF.

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676–684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166±26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.

Annexins are a family of 13 proteins known to bind phospholipids (PL) in a Ca(2+)-dependent way. They are ubiquitous proteins and share a similar structure characterized by a conserved C-terminal domain with Ca(2+) binding sites and a variable N-terminal domain. Depending on Ca(2+) concentration, they have been reported to participate in a variety of membrane-related events such as exocytosis, endocytosis, apoptosis and binding to cytoskeletal proteins. They have also been reported to regulate protein activities. This review will focus on annexins in the heart, and particularly on annexins A2, A5, A6 and A7. Annexin A2 has been found in endothelial cells and reported to play a central role in control of plasmin-mediated processes. Annexin A5 is mainly localized in cardiomyocytes. However, it could be relocated to interstitial tissue in ischemic and failing hearts or it could be externalized and exhibit a proapoptotic effect in cardiomyocytes. Annexin A6 is the most abundant annexin in the heart, and has been localized in various cell types including myocytes. Overexpression of annexin A6 has underlined physiological alterations in contractile mechanics leading to dilated cardiomyopathy, whereas knockout has been found to induce faster changes in Ca(2+) transient and increased contractility, suggesting a negative inotropic role for annexin A6. Annexin A7 is expressed in heart and skeletal muscle. In annexin A7 null mutant mice decreases in the force-frequency relationship were observed in adult cardiomyocytes, consistent with regulation of Ca(2+) handling. In conclusion, while annexin A2 was involved in regulation of fibrin homeostasis, alterations in expression and activity of annexins A5, A6 and A7 have been associated with regulation of Ca(2+) handling in the heart, but the target of each annexin has not yet been identified.

The medical options available to prevent or treat radiation-induced injury are scarce and developing effective countermeasures is still an open research field. In addition, more than half of cancer patients are treated with radiation therapy, which displays a high antitumor efficacy but can cause, albeit rarely, disabling long-term toxicities including radiation fibrosis. Progress has been made in the definition of molecular pathways associated with normal tissue toxicity that suggest potentially effective therapeutic targets. Targeting the Rho/ROCK pathway seems a promising anti-fibrotic approach, at least in the gut; the current study was performed to assess whether this target was relevant to the prevention and/or treatment of injury to the main thoracic organs, namely heart and lungs. First, we showed activation of two important fibrogenic pathways (Smad and Rho/ROCK) in response to radiationexposure to adult cardiomyocytes; we extended these observations in vivo to the heart and lungs of mice, 15 and 30 weeks post-irradiation. We correlated this fibrogenic molecular imprint with alteration of heart physiology and long-term remodelling of pulmonary and cardiac histological structures. Lastly, cardiac and pulmonary radiation injury and bleomycin-induced pulmonary fibrosis were successfully modulated using Rho/ROCK inhibitors (statins and Y-27632) and this was associated with a normalization of fibrogenic markers. In conclusion, the present paper shows for the first time, activation of Rho/ROCK and Smad pathways in pulmonary and cardiac radiation-induced delayed injury. Our findings thereby reveal a safe and efficient therapeutic opportunity for the abrogation of late thoracic radiation injury, potentially usable either before or after radiation exposure; this approach is especially attractive in (i) the radiation oncology setting, as it does not interfere with prior anti-cancer treatment and in (ii) radioprotection, as applicable to the treatment of established radiation injury, for example in the case of radiation accidents or acts of terrorism.