Rhona O'leary

Cardiotrophin-1 (CT-1) can protect the adult heart from injury when added both prior to ischaemia and at reperfusion

OBJECTIVES To determine whether the cytokine cardiotrophin-1 (CT-1) can protect the adult heart against ischaemia/reperfusion when added either prior to ischaemia or at reperfusion. BACKGROUND CT-1 has previously been shown to protect cultured embryonic or neonatal cardiocytes from cell death. To assess the therapeutic potential of CT-1, it is necessary to determine whether this effect can be observed in adult cardiac cells both in culture and most importantly in the intact heart. METHODS We examined the protective effect of CT-1 both in cultured adult rat cardiocytes and in the rat intact heart. In both cases, the cardiac cells were exposed to hypoxia/ischaemia followed by reoxygenation/reperfusion and CT-1 was administered either prior to hypoxia/ischaemia or at reoxygenation/reperfusion. RESULTS CT-1 has a protective effect in reducing ischaemic damage in the intact heart ex vivo as assayed by infarct size to area at risk ratio (20% compared to 35%). Similar protective effects against cell death were noted in adult cells in vitro. Both in vitro and ex vivo CT-1 can exert a protective effect when added at the time of reoxygenation/reperfusion as well as prior to the hypoxic/ischaemic stimulus (cell death reduced from 50 to 20% in TUNEL assay, infarct size to zone at risk ratio reduced from 35 to 20%). These protective effects are blocked by an inhibitor of the p42/p44 MAPK pathway. CONCLUSION CT-1 can protect adult cardiac cells both in vitro and in vivo when added both prior to or after the hypoxic/ischaemic stimulus. The potential therapeutic benefit of CT-1 when added at the time of reperfusion following ischaemic damage is discussed.

Cardiotrophin-1 can protect cardiac myocytes from injury when added both prior to simulated ischaemia and at reoxygenation

OBJECTIVE The cytokine cardiotrophin-1 (CT-1) has previously been shown to protect cultured cardiocytes from cell death induced by serum removal or hypoxia when administered prior to the damaging stimulus. We wished to test whether a similar protective effect could be observed if CT-1 was added after the ischaemic period and to investigate the signalling pathways involved in the protective effect when CT-1 is given prior to or after ischaemia. METHODS We therefore examined the protective effect of CT-1 in cultured rat cardiocytes exposed to simulated ischaemia followed by reoxygenation when CT-1 was administered either prior to simulated ischaemia or at reoxygenation. RESULTS We show that CT-1 can exert a protective effect against the damaging effects of simulated ischaemia/reoxygenation both when added after the simulated ischaemia at reoxygenation (P<0.05 in trypan blue, TUNEL and annexin V assays) or when added prior to the simulated ischaemia (P<0.05). In both cases, these protective effects are blocked by an inhibitor of the p42/p44 MAPK pathway (P<0.05 in all assays). CONCLUSION CT-1 can protect cardiac cells when added either prior to simulated ischaemia or at the time of reoxygenation following simulated ischaemia and these effects are dependent upon its ability to activate the p42/p44 MAPK pathway. Hence CT-1 may have therapeutic potential when added at the time of reperfusion following ischaemic damage.

Brain‐Derived Neurotrophic Factor Induces Excitotoxic Sensitivity in Cultured Embryonic Rat Spinal Motor Neurons Through Activation of the Phosphatidylinositol 3‐Kinase Pathway

Abstract: Neurotrophic factors (NTFs) can protect against or sensitize neurons to excitotoxicity. We studied the role played by various NTFs in the excitotoxic death of purified embryonic rat motor neurons. Motor neurons cultured in brain‐derived neurotrophic factor, but not neurotrophin 3, glial‐derived neurotrophic factor, or cardiotrophin 1, were sensitive to excitotoxic insult. BDNF also induces excitotoxic sensitivity (ES) in motor neurons when BDNF is combined with these other NTFs. The effect of BDNF depends on de novo protein and mRNA synthesis. Reagents that either activate or inhibit the 75‐kDa NTF receptor p75NTR do not affect BDNF‐induced ES. The low EC50 for BDNF‐induced survival and ES suggests that TrkB mediates both of these biological activities. BDNF does not alter glutamate‐evoked rises of intracellular Ca2+, suggesting BDNF acts downstream. Both wortmannin and LY294002, which specifically block the phosphatidylinositol 3‐kinase (PI3K) intracellular signaling pathway in motor neurons, inhibit BDNF‐induced ES. We confirm this finding using a herpes simplex virus (HSV) that expresses the dominant negative p85 subunit of PI3K. Infecting motor neurons with this HSV, but not a control HSV, blocks activation of the PI3K pathway and BDNF‐induced ES. Through the activation of TrkB and the PI3K signaling pathway, BDNF renders developing motor neurons susceptible to glutamate receptor‐mediated cell death.

Effects of cardiotrophin‐1 (CT‐1) in a mouse motor neuron disease

Cardiotrophin‐1 (CT‐1) has potent survival‐promoting effects on motor neurons in vitro and in vivo and may be effective in treating motor neuron diseases (MND). We investigated the effects of CT‐1 treatment in wobbler mouse MND. Wobbler mice were randomly assigned to receive subcutaneously injected CT‐1 (1 mg/kg, n = 18, in two experiments) or vehicle (n = 18, in two experiments) daily, 6 times/week for 4 weeks after clinical diagnosis at age 3 to 4 weeks. Cardiotrophin‐1 treatment prevented deterioration in paw position and walking pattern abnormalities. Grip strength declined steadily in the vehicle group, whereas in the CT‐1 group it declined at week 1 but increased thereafter to exceed baseline strength by 5% (P = 0.0002) at week 4. Running speed was faster with CT‐1 (P = 0.007). Biceps muscle twitch tension, muscle weight, mean muscle fiber diameter, and intramuscular axonal sprouting were significantly greater with CT‐1 treatment than with vehicle treatment. Histometry revealed a trend that indicated CT‐1 modestly increased the number of immunoreactive motor neurons, as determined by both choline acetyltransferase and c‐Ret antibodies, and reduced the number of phosphorylated neurofilament immunoreactive perikarya (P = 0.05). The number of large myelinated motor axons significantly increased with treatment (206 versus 113, P = 0.01). We conclude that CT‐1 exerts myotrophic effects as well as neurotrophic effects in a mouse model of spontaneous MND, a finding that has potential therapeutic implications for human MND.