No abstract

In mammalian cardiac ventricular myocytes, Ca influx and release occur predominantly at t-tubules, ensuring synchronous Ca release throughout the cell. Heart failure is associated with disrupted t-tubule structure, but its effect on t-tubule function is less clear. We therefore investigated Ca influx and release at the t-tubules of ventricular myocytes isolated from rat hearts ~ 18 weeks after coronary artery ligation (CAL) or corresponding Sham operation. L-type Ca current (ICa) was recorded using the whole-cell voltage-clamp technique in intact and detubulated myocytes; Ca release at t-tubules was monitored using confocal microscopy with voltage- and Ca-sensitive fluorophores. CAL was associated with cardiac and cellular hypertrophy, decreased ejection fraction, disruption of t-tubule structure and a smaller, slower Ca transient, but no change in ryanodine receptor distribution, L-type Ca channel expression, or ICa density. In Sham myocytes, ICa was located predominantly at the t-tubules, while in CAL myocytes, it was uniformly distributed between the t-tubule and surface membranes. Inhibition of protein kinase A with H-89 caused a greater decrease of t-tubular ICa in CAL than in Sham myocytes; in the presence of H-89, t-tubular ICa density was smaller in CAL than in Sham myocytes. The smaller t-tubular ICa in CAL myocytes was accompanied by increased latency and heterogeneity of SR Ca release at t-tubules, which could be mimicked by decreasing ICa using nifedipine. These data show that CAL decreases t-tubular ICa via a PKA-independent mechanism, thereby impairing Ca release at t-tubules and contributing to the altered excitation–contraction coupling observed in heart failure.

Besides being essential for correct development of the vertebrate nervous system the neurotrophins also play a vital role in adult neuron survival, maintenance and regeneration. In addition they are implicated in the pathogenesis of certain neurodegenerative diseases, and may even provide a therapeutic solution for some. In particular there have been a number of studies on the involvement of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) in the development of Alzheimer’s disease. This disease is of growing concern as longevity increases worldwide, with little treatment available at the moment to alleviate the condition. Memory loss is one of the earliest symptoms associated with Alzheimer’s disease. The brain regions first affected by pathology include the hippocampus, and also the entorhinal cortex and basal cholinergic nuclei which project to the hippocampus; importantly, all these areas are required for memory formation. Both NGF and BDNF are affected early in the disease and this is thought to initiate a cascade of events which exacerbates pathology and leads to the symptoms of dementia. This review briefly describes the pathology, symptoms and molecular processes associated with Alzheimer’s disease; it discusses the involvement of the neurotrophins, particularly NGF and BDNF, and their receptors, with changes in BDNF considered particularly in the light of its importance in synaptic plasticity. In addition, the possibilities of neurotrophin-based therapeutics are evaluated.

L-type Ca channels (LTCC), which play a key role in cardiac excitation–contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation.

Nerve growth factor (NGF) promotes cell survival via binding to the tyrosine kinase receptor A (TrkA). Its precursor, proNGF, binds to p75NTR and sortilin receptors to initiate apoptosis. Current disagreement exists over whether proNGF acts neurotrophically following binding to TrkA. As in Alzheimer’s disease the levels of proNGF increase and TrkA decrease, it is important to clarify the properties of proNGF. Here, wild‐type and cleavage‐resistant mutated forms (M) of proNGF were engineered and their binding characteristics determined. M‐proNGF and NGF bound to p75NTR with similar affinities, whilst M‐proNGF had a lower affinity than NGF for TrkA. M‐proNGF behaved neurotrophically, albeit less effectively than NGF. M‐proNGF addition resulted in phosphorylation of TrkA and ERK1/2, and in PC12 cells elicited neurite outgrowth and supported cell survival. Conversely, M‐proNGF addition to cultured cortical neurons initiated caspase 3 cleavage. Importantly, these biological effects were shown to be mediated by unprocessed M‐proNGF. Surprisingly, binding of the pro region alone to TrkA, at a site other than that of NGF, caused TrkA and ERK1/2 phosphorylation. Our data show that M‐proNGF stimulates TrkA to a lesser degree than NGF, suggesting that in Alzheimer brain the increased proNGF : NGF and p75NTR : TrkA ratios may permit apoptotic effects to predominate over neurotrophic effects.

Chronic pain presents a huge economic and social burden, with existing treatments largely unable to satisfy medical needs. Recently, studies have shown that nerve growth factor (NGF) is a major mediator of inflammatory and neuropathic pain, providing a new therapeutic target. Although originally discovered as a trophic factor for sympathetic and sensory neurons during development, it now appears that in adults, levels of NGF are elevated in many acute and chronic pain conditions. Furthermore, preclinical animal models of inflammatory and neuropathic pain also show increased NGF levels, while the sequestration of NGF alleviates the associated hyperalgesia. The molecular mechanisms involved are being elucidated. This review briefly examines pain signaling pathways and describes currently available analgesics. It then investigates the approaches taken in targeting NGF-mediated pain. Current options being explored include the development of humanized monoclonal antibodies to NGF or its tyrosine kinase receptor TrkA (also known as neurotrophic tyrosine kinase receptor, type 1 [NTRK1]), and the sequestration of NGF using TrkA domain 5 (TrkAd5), a soluble receptor protein that binds NGF with picomolar affinity. Administration of either antibodies or TrkAd5 has been shown to be effective in a number of preclinical models of pain, including cystitis, osteoarthritis, UV irradiation (sunburn), and skeletal bone pain due to fracture or cancer. Other possible future therapies examined in this review include small-molecule TrkA antagonists, which target either the extracellular NGF binding domain of TrkA or its intracellular tyrosine kinase domain.

New Findings What is the central question of this study? Heart failure is associated with redistribution of L‐type Ca2+ current (I Ca) away from the t‐tubule membrane to the surface membrane of cardiac ventricular myocytes. However, the underlying mechanism and its dependence on severity of pathology (hypertrophy versus failure) are unclear. What is the main finding and its importance? Increasing severity of response to transverse aortic constriction, from hypertrophy to failure, was accompanied by graded loss of t‐tubular I Ca and loss of regulation of I Ca by caveolin 3. Thus, the pathological loss of t‐tubular I Ca, which contributes to impaired excitation–contraction coupling and thereby cardiac function in vivo, appears to be attributable to loss of caveolin 3‐dependent stimulation of t‐tubular I Ca. AbstractPrevious work has shown redistribution of L‐type Ca2+ current (I Ca) from the t‐tubules to the surface membrane of rat ventricular myocytes after myocardial infarction. However, whether this occurs in all species and in response to other insults, the relationship of this redistribution to the severity of the pathology, and the underlying mechanism, are unknown. We have therefore investigated the response of mouse hearts and myocytes to pressure overload induced by transverse aortic constriction (TAC). Male C57BL/6 mice underwent TAC or equivalent sham operation 8 weeks before use. I Ca and Ca2+ transients were measured in isolated myocytes, and expression of caveolin 3 (Cav3), junctophilin 2 (Jph2) and bridging integrator 1 (Bin1) was determined. C3SD peptide was used to disrupt Cav3 binding to its protein partners. Some animals showed cardiac hypertrophy in response to TAC with little evidence of heart failure, whereas others showed greater hypertrophy and pulmonary congestion. These graded changes were accompanied by graded cellular hypertrophy, t‐tubule disruption, decreased expression of Jph2 and Cav3, and decreased t‐tubular I Ca density, with no change at the cell surface, and graded impairment of Ca2+ release at t‐tubules. C3SD decreased I Ca density in control but not in TAC myocytes. These data suggest that the graded changes in cardiac function and size that occur in response to TAC are paralleled by graded changes in cell structure and function, which will contribute to the impaired function observed in vivo. They also suggest that loss of t‐tubular I Ca is attributable to loss of Cav3‐dependent stimulation of I Ca.

Aging is associated with diminished cardiac function in males. Cardiac excitation-contraction coupling in ventricular myocytes involves Ca influx via the Ca current (ICa) and Ca release from the sarcoplasmic reticulum, which occur predominantly at t-tubules. Caveolin-3 regulates t-tubular ICa, partly through protein kinase A (PKA), and both ICa and caveolin-3 decrease with age. We therefore investigated ICa and t-tubule structure and function in cardiomyocytes from male wild-type (WT) and caveolin-3-overexpressing (Cav-3OE) mice at 3 and 24 months of age. In WT cardiomyocytes, t-tubular ICa-density was reduced by ~50% with age while surface ICa density was unchanged. Although regulation by PKA was unaffected by age, inhibition of caveolin-3-binding reduced t-tubular ICa at 3 months, but not at 24 months. While Cav-3OE increased cardiac caveolin-3 protein expression ~2.5-fold at both ages, the age-dependent reduction in caveolin-3 (WT ~35%) was preserved in transgenic mice. Overexpression of caveolin-3 reduced t-tubular ICa density at 3 months but prevented further ICa loss with age. Measurement of Ca release at the t-tubules revealed that the triggering of local Ca release by t-tubular ICa was unaffected by age. In conclusion, the data suggest that the reduction in ICa density with age is associated with the loss of a caveolin-3-dependent mechanism that augments t-tubular ICa density.