Elimination of fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure.

Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.

TRPV2 is required for mechanical nociception and the stretch-evoked response of primary sensory neurons.

Mechanotransduction plays important roles in many sensory processes, including touch, pain, hearing, and proprioception. However, the molecular mechanisms of mechanical nociception have remained unclear. Here, we showed that elimination of transient receptor potential vanilloid 2 (TRPV2) in mice resulted in the deficit of mechanical nociception due to the lack of mechanosensitivity in a subclass of adult primary sensory neurons (PSNs). The PSN-specific TRPV2-deficient mice showed behavioural impairment of mechanical nociception in tail-pressure and von Frey hair tests, without defects in axonal growth and neuronal composition. Conversely, the mice displayed normal behaviour to noxious heat and non-noxious tactile stimuli. Furthermore, based on the stretch-evoked Ca2+ response of cultured PSNs, we characterised two types of stretch-activated neurons in normal mice; fast-decay high-threshold and slow-decay low-threshold mechanosensitive. The cultured neurons from TRPV2-deficient mice lacked stretch-evoked Ca2+ responses by fast-decay neurons normally activated by high-threshold mechanical stimulation. These results demonstrated that TRPV2 has a critical role in mechanical nociception in the adult somatosensory system.

Moderate intensity, exercise-induced catecholamine release in the preoptic area and anterior hypothalamus in rats is enhanced in a warm environment.

Thermoeffector responses and core body temperature (Tcore) homeostasis during exercise are affected by both ambient temperature and exercise intensity. We have previously reported that Tcore, heat loss responses, and catecholamine release in the preoptic area and anterior hypothalamus (PO/AH) increased during incremental treadmill running. However, no previous study has examined whether changes in the thermoregulatory responses at warm ambient temperature are related to catecholamine responses during moderate intensity exercise in rats. Therefore, the aim of the present study was to investigate the responsiveness of neurotransmission in the PO/AH to moderate intensity exercise at different ambient temperatures, and to relate this to changes in thermoregulation. We measured the monoamine levels in the PO/AH and the thermoregulatory responses in exercising rats simultaneously using a combination of methods, including in vivo microdialysis, biotelemetry, and animal O2/CO2 metabolism measuring system. On the day of experiments, rats ran for 60min at a speed of 18mmin-1 on a treadmill at a 5% gradient, in an ambient temperature of 23°C or 30°C. Tcore, tail skin temperature (Ttail; an index of heat loss), and oxygen consumption (V̇O2: an index of heat production) were monitored. Dopamine (DA), noradrenaline (NA), and serotonin (5-HT) levels were measured by high performance liquid chromatography (HPLC) with electrochemical detection. Exercise significantly increased the Tcore, Ttail, and V̇O2 values, as well as DA and NA release in the PO/AH at both temperatures, and the increases were more pronounced at the warm ambient temperature. The results suggest that the increase in the Tcore, heat production, and heat loss responses even during moderate intensity running in a warm environment are likely associated with an increase in DA and NA release in the PO/AH region.

Induced NCX1 overexpression attenuates pressure overload-induced pathological cardiac remodelling.

AIMS: Although increased Na(+)/Ca(2+) exchanger 1 (NCX1) expression is observed during heart failure (HF), the pathological role of NCX1 during the progression of HF remains unclear. We examined alterations of NCX1 expression and activity in hearts after transverse aortic constriction (TAC) surgery and explored whether NCX1 influences pressure overload-induced pathological cardiac remodelling. METHODS AND RESULTS: We generated novel transgenic mice in which NCX1 expression is controlled by a cardiac-specific, doxycycline (DOX)-dependent promoter. In the absence of DOX, TAC surgery caused substantial chamber dilation with a gradual decrease in contractility by 16 weeks. Cardiomyocytes showed a decline in contractility with abnormal Ca(2+) handling during excitation-contraction (E-C) coupling. Reduced NCX1 activity was observed 8 weeks after TAC and was still apparent at 17 weeks. Induced NCX1 overexpression by DOX treatment starting 8 weeks after TAC returned NCX1 activity to pre-TAC levels and prevented chamber dilation with cardiac dysfunction. DOX treatment not only upregulated NCX1 expression in TAC-operated hearts but also returned L-type Ca(2+) channel and sarcoplasmic reticulum (SR) Ca(2+) ATPase expression levels to those in sham-operated hearts. In DOX-treated myocytes, contractility, T-tubule integrity, synchrony of Ca(2+) release from the SR, and Ca(2+) handling during E-C coupling was preserved 16 weeks after TAC surgery. In addition, DOX treatment attenuated the down-regulation of survival signalling and up-regulation of apoptosis signalling 16 weeks after TAC surgery. CONCLUSION: Induced overexpression of NCX1 attenuated pressure overload-induced pathological cardiac remodelling. Thus, maintaining NCX1 activity may be a potential therapeutic strategy for preventing the progression of HF.

TRPV2 is critical for the maintenance of cardiac structure and function in mice.

The heart has a dynamic compensatory mechanism for haemodynamic stress. However, the molecular details of how mechanical forces are transduced in the heart are unclear. Here we show that the transient receptor potential, vanilloid family type 2 (TRPV2) cation channel is critical for the maintenance of cardiac structure and function. Within 4 days of eliminating TRPV2 from hearts of the adult mice, cardiac function declines severely, with disorganization of the intercalated discs that support mechanical coupling with neighbouring myocytes and myocardial conduction defects. After 9 days, cell shortening and Ca(2+) handling by single myocytes are impaired in TRPV2-deficient hearts. TRPV2-deficient neonatal cardiomyocytes form no intercalated discs and show no extracellular Ca(2+)-dependent intracellular Ca(2+) increase and insulin-like growth factor (IGF-1) secretion in response to stretch stimulation. We further demonstrate that IGF-1 receptor/PI3K/Akt pathway signalling is significantly downregulated in TRPV2-deficient hearts, and that IGF-1 administration partially prevents chamber dilation and impairment in cardiac pump function in these hearts. Our results improve our understanding of the molecular processes underlying the maintenance of cardiac structure and function.

Acute intraperitoneal injection of caffeine improves endurance exercise performance in association with increasing brain dopamine release during exercise.

The purpose of this study was to examine changes of thermoregulation, neurotransmitters in the preoptic area and anterior hypothalamus (PO/AH), which is the thermoregulatory center, and endurance exercise performance after the intraperitoneal injection of caffeine in rats. Core body temperature (Tcore), oxygen consumption (VO2) and tail skin temperature (Ttail) were measured. A microdialysis probe was inserted in the PO/AH, and samples for the measurements of extracellular dopamine (DA), noradrenaline (NA) and serotonin (5-HT) levels were collected. During the rest experiment, 1 h after baseline collections in the chamber (23 °C), the rats were intraperitoneally injected with saline, or 3 mg kg(-1) or 10 mg kg(-1) caffeine. The duration of the test was 4 h. During the exercise experiment, baseline collections on the treadmill were obtained for 1 h. One hour before the start of exercise, rats were intraperitoneally injected with either 10 mg kg(-1) caffeine (CAF) or saline (SAL). Animals ran until fatigue at a speed of 18 m min(-1), at a 5% grade, on the treadmill in a normal environment (23 °C). At rest, 3 mg kg(-1) caffeine did not influence Tcore, Ttail, VO2, extracellular DA, NA and 5-HT. 10 mg kg(-1) caffeine caused significant increases in Tcore, VO2, Ttail and extracellular DA in the PO/AH. In addition, 10 mg kg(-1) caffeine increased the run time to fatigue (SAL: 104.4 ± 30.9 min, CAF: 134.0 ± 31.1 min, p<0.05). The combination of caffeine and exercise increased Tcore, VO2, Ttail and extracellular DA in the PO/AH. NA increased during exercise, while neither caffeine nor exercise changed 5-HT. These results indicate that caffeine has ergogenic and hyperthermic effects, and these effects may be related to changes of DA release in the brain.

Continuous monitoring of hypothalamic neurotransmitters and thermoregulatory responses in exercising rats.

The purpose of the present study was to investigate the relationship between thermoregulation and catecholamine release in the preoptic area and anterior hypothalamus (PO/AH) during incremental treadmill running in the rat. To this aim, we combined in vivo brain microdialysis, biotelemetry and metabolic measurements for continuous monitoring of core body temperature (T(core)), neurotransmitters and thermoregulatory responses. The animals were exercised for 1h at 23°C. Treadmill speed was increased every 20 min (10, 20 and 26 m min⁻¹). T(core), oxygen consumption (V˙O2, an index of heat production) and tail skin temperature (T(tail), an index of heat loss) were simultaneously measured. Brain microdialysis samples were collected every 10 min, and these samples were analyzed for noradrenaline (NA), dopamine (DA) and serotonin (5-HT). T(core) and V˙O(2)2 significantly increased during treadmill and were exercise intensity dependent. After an initial drop T(tail) increased significantly during exercise. Both NA and DA levels in the PO/AH increased significantly during exercise. There was no effect on serotonin release. T(core), V˙O2 and T(tail) were positively correlated with the levels of NA and DA. Our data suggest that thermoregulatory responses are dependent on the intensity of the exercise and that these responses are associated with changes in NA and DA release, but not in 5-HT release in the PO/AH.

Serotonin release in the preoptic area and anterior hypothalamus is not involved in thermoregulation during low-intensity exercise in a warm environment.

The aim of the present study was to assess the response of serotonin (5-hydroxytryptamine; 5-HT) in the preoptic area and anterior hypothalamus (PO/AH) to thermoregulation during exercise in a warm environment. In addition, it was investigated whether a rapid increase in extracellular 5-HT levels in the PO/AH modifies the thermoregulatory response under the same exercise conditions. Rats were made to run for 120 min at 10 m min(-1) on a treadmill at the ambient temperature of 30 degrees C. Body core temperature (T(core)) was monitored using a biotelemetry system, and tail skin temperature (T(tail)) was simultaneously measured as an index of heat loss response. Microdialysis in combination with HPLC was used to measure concentrations of monoamines in the PO/AH. Both T(core) and T(tail) increased during the first 20 min of exercise and remained stable until the end of the exercise period. Low-intensity exercise did not induce any changes in 5-HT release in the PO/AH, although the levels of norepinephrine and dopamine were increased. Moreover, increased extracellular 5-HT by local perfusion of 1 microM citalopram (selective 5-HT reuptake inhibitor; SSRI) in the PO/AH had no effect on the thermoregulatory response during acute low-intensity exercise in a warm environment. These results suggest that enhanced release of only 5-HT in the PO/AH may not intervene thermoregulation during exercise in a warm environment.