Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease.

Currently there is an unmet need for treatments that can prevent hypertrophic cardiomyopathy (HCM). Using a murine model we previously identified that HCM causing cardiac troponin I mutation Gly203Ser (cTnI-G203S) is associated with increased mitochondrial metabolic activity, consistent with the human condition. These alterations precede development of the cardiomyopathy. Here we examine the efficacy of in vivo treatment of cTnI-G203S mice with a peptide derived against the α-interaction domain of the cardiac L-type calcium channel (AID-TAT) on restoring mitochondrial metabolic activity, and preventing HCM. cTnI-G203S or age-matched wt mice were treated with active or inactive AID-TAT. Following treatment, targeted metabolomics was utilized to evaluate myocardial substrate metabolism. Cardiac myocyte mitochondrial metabolic activity was assessed as alterations in mitochondrial membrane potential and flavoprotein oxidation. Cardiac morphology and function were examined using echocardiography. Cardiac uptake was assessed using an in vivo multispectral imaging system. We identified alterations in six biochemical intermediates in cTnI-G203S hearts consistent with increased anaplerosis. We also reveal that AID-TAT treatment of precardiomyopathic cTnI-G203S mice, but not mice with established cardiomyopathy, restored cardiac myocyte mitochondrial membrane potential and flavoprotein oxidation, and prevented myocardial hypertrophy. Importantly, AID-TAT was rapidly targeted to the heart, and not retained by the liver or kidneys. Overall, we identify biomarkers of HCM resulting from the cTnI mutation Gly203Ser, and present a safe, preventative therapy for associated cardiomyopathy. Utilizing AID-TAT to modulate cardiac metabolic activity may be beneficial in preventing HCM in "at risk" patients with identified Gly203Ser gene mutations.

Glutathionylation of the L-type Ca2+ channel in oxidative stress-induced pathology of the heart.

There is mounting evidence to suggest that protein glutathionylation is a key process contributing to the development of pathology. Glutathionylation occurs as a result of posttranslational modification of a protein and involves the addition of a glutathione moiety at cysteine residues. Such modification can occur on a number of proteins, and exerts a variety of functional consequences. The L-type Ca2+ channel has been identified as a glutathionylation target that participates in the development of cardiac pathology. Ca2+ influx via the L-type Ca2+ channel increases production of mitochondrial reactive oxygen species (ROS) in cardiomyocytes during periods of oxidative stress. This induces a persistent increase in channel open probability, and the resulting constitutive increase in Ca2+ influx amplifies the cross-talk between the mitochondria and the channel. Novel strategies utilising targeted peptide delivery to uncouple mitochondrial ROS and Ca2+ flux via the L-type Ca2+ channel following ischemia-reperfusion have delivered promising results, and have proven capable of restoring appropriate mitochondrial function in myocytes and in vivo.

A protein synthesis and nitric oxide-dependent presynaptic enhancement in persistent forms of long-term potentiation.

Long-term potentiation (LTP) is an important process underlying learning and memory in the brain. At CA3-CA1 synapses in the hippocampus, three discrete forms of LTP (LTP1, 2, and 3) can be differentiated on the basis of maintenance and induction mechanisms. However, the relative roles of pre- and post-synaptic expression mechanisms in LTP1, 2, and 3 are unknown. Neurotransmitter release in the expression of LTP1, 2, and 3 was measured via FM 1-43 destaining from CA3 terminals in hippocampal slices from male Wistar rats (7-8 wk). No difference in vesicle turnover rate was observed for LTP1 up to 160 min following induction by one train of theta-burst stimulation (1TBS). A presynaptic enhancement was found for LTP2 at 160 min after induction by 4TBS, and for LTP3 at both 80 and 160 min after induction by 8TBS. Inhibition of nitric oxide (NO) signaling blocked both LTP2 and LTP3 maintenance and the associated enhanced release. LTP2 maintenance and its presynaptic expression were dependent on protein synthesis, but not gene transcription. LTP3 maintenance was dependent on both translation and transcription, but like LTP2, the enhanced release only required translation. These data considerably strengthen the mechanistic separation of LTP1, 2, and 3, supporting a model of multiple, discrete forms of LTP at CA3-CA1 synapses rather than different temporal phases.

A Morpholino Oligomer Therapy Regime That Restores Mitochondrial Function and Prevents mdx Cardiomyopathy.

Current clinical trials demonstrate Duchenne muscular dystrophy (DMD) patients receiving phosphorodiamidate morpholino oligomer (PMO) therapy exhibit improved ambulation and stable pulmonary function; however, cardiac abnormalities remain. Utilizing the same PMO chemistry as current clinical trials, we have identified a non-toxic PMO treatment regimen that restores metabolic activity and prevents DMD cardiomyopathy. We propose that a treatment regimen of this nature may have the potential to significantly improve morbidity and mortality from DMD by improving ambulation, stabilizing pulmonary function, and preventing the development of cardiomyopathy.

Dystrophic Cardiomyopathy-Potential Role of Calcium in Pathogenesis, Treatment and Novel Therapies.

Duchenne muscular dystrophy (DMD) is caused by defects in the DMD gene and results in progressive wasting of skeletal and cardiac muscle due to an absence of functional dystrophin. Cardiomyopathy is prominent in DMD patients, and contributes significantly to mortality. This is particularly true following respiratory interventions that reduce death rate and increase ambulation and consequently cardiac load. Cardiomyopathy shows an increasing prevalence with age and disease progression, and over 95% of patients exhibit dilated cardiomyopathy by the time they reach adulthood. Development of the myopathy is complex, and elevations in intracellular calcium, functional muscle ischemia, and mitochondrial dysfunction characterise the pathophysiology. Current therapies are limited to treating symptoms of the disease and there is therefore an urgent need to treat the underlying genetic defect. Several novel therapies are outlined here, and the unprecedented success of phosphorodiamidate morpholino oligomers (PMOs) in preclinical and clinical studies is overviewed.

Progesterone Sharpens Temporal Response Profiles of Sensory Cortical Neurons in Animals Exposed to Traumatic Brain Injury.

Traumatic brain injury (TBI) initiates a cascade of pathophysiological changes that are both complex and difficult to treat. Progesterone (P4) is a neuroprotective treatment option that has shown excellent preclinical benefits in the treatment of TBI, but these benefits have not translated well in the clinic. We have previously shown that P4 exacerbates the already hypoactive upper cortical responses in the short-term post-TBI and does not reduce upper cortical hyperactivity in the long term, and we concluded that there is no tangible benefit to sensory cortex firing strength. Here we examined the effects of P4 treatment on temporal coding resolution in the rodent sensory cortex in both the short term (4 d) and long term (8 wk) following impact-acceleration-induced TBI. We show that in the short-term postinjury, TBI has no effect on sensory cortex temporal resolution and that P4 also sharpens the response profile in all cortical layers in the uninjured brain and all layers other than layer 2 (L2) in the injured brain. In the long term, TBI broadens the response profile in all cortical layers despite firing rate hyperactivity being localized to upper cortical layers and P4 sharpens the response profile in TBI animals in all layers other than L2 and has no long-term effect in the sham brain. These results indicate that P4 has long-term effects on sensory coding that may translate to beneficial perceptual outcomes. The effects seen here, combined with previous beneficial preclinical data, emphasize that P4 is still a potential treatment option in ameliorating TBI-induced disorders.

Adult-onset obesity is triggered by impaired mitochondrial gene expression.

Mitochondrial gene expression is essential for energy production; however, an understanding of how it can influence physiology and metabolism is lacking. Several proteins from the pentatricopeptide repeat (PPR) family are essential for the regulation of mitochondrial gene expression, but the functions of the remaining members of this family are poorly understood. We created knockout mice to investigate the role of the PPR domain 1 (PTCD1) protein and show that loss of PTCD1 is embryonic lethal, whereas haploinsufficient, heterozygous mice develop age-induced obesity. The molecular defects and metabolic consequences of mitochondrial protein haploinsufficiency in vivo have not been investigated previously. We show that PTCD1 haploinsufficiency results in increased RNA metabolism, in response to decreased protein synthesis and impaired RNA processing that affect the biogenesis of the respiratory chain, causing mild uncoupling and changes in mitochondrial morphology. We demonstrate that with age, these effects lead to adult-onset obesity that results in liver steatosis and cardiac hypertrophy in response to tissue-specific differential regulation of the mammalian target of rapamycin pathways. Our findings indicate that changes in mitochondrial gene expression have long-term consequences on energy metabolism, providing evidence that haploinsufficiency of PTCD1 can be a major predisposing factor for the development of metabolic syndrome.

Progesterone Exacerbates Short-Term Effects of Traumatic Brain Injury on Supragranular Responses in Sensory Cortex and Over-Excites Infragranular Responses in the Long Term.

Progesterone (P4) has been suggested as a neuroprotective agent for traumatic brain injury (TBI) because it ameliorates many post-TBI sequelae. We examined the effects of P4 treatment on the short-term (4 days post-TBI) and long-term (8 weeks post-TBI) aftermath on neuronal processing in the rodent sensory cortex of impact acceleration-induced diffuse TBI. We have previously reported that in sensory cortex, diffuse TBI induces a short-term hypoexcitation that is greatest in the supragranular layers and decreases with depth, but a long-term hyperexcitation that is exclusive to the supragranular layers. Now, adult male TBI-treated rats administered P4 showed, in the short term, even greater suppression in neural responses in supragranular layers but a reversal of the TBI-induced suppression in granular and infragranular layers. In long-term TBI there were only inconsistent effects of P4 on the TBI-induced hyperexcitation in supragranular responses but infragranular responses, which were not affected by TBI alone, were elevated by P4 treatment. Intriguingly, the effects in the injured brain were almost identical to P4 effects in the normal brain, as seen in sham control animals treated with P4: in the short term, P4 effects in the normal brain were identical to those exercised in the injured brain and in the long term, P4 effects in the normal brain were rather similar to what was seen in the TBI brain. Overall, these results provide no support for any protective effects of P4 treatment on neuronal encoding in diffuse TBI, and this was reflected in sensorimotor and other behavior tasks also tested here. Additionally, the effects suggest that mechanisms used for P4 effects in the normal brain are also intact in the injured brain.

The Role of the L-Type Ca2+ Channel in Altered Metabolic Activity in a Murine Model of Hypertrophic Cardiomyopathy.

Heterozygous mice (αMHC403/+ ) expressing the human disease-causing mutation Arg403Gln exhibit cardinal features of hypertrophic cardiomyopathy (HCM) including hypertrophy, myocyte disarray, and increased myocardial fibrosis. Treatment of αMHC403/+ mice with the L-type calcium channel (ICa-L) antagonist diltiazem has been shown to decrease left ventricular anterior wall thickness, cardiac myocyte hypertrophy, disarray, and fibrosis. However, the role of the ICa-L in the development of HCM is not known. In addition to maintaining cardiac excitation and contraction in myocytes, the ICa-L also regulates mitochondrial function through transmission of movement of ICa-L via cytoskeletal proteins to mitochondrial voltage-dependent anion channel. Here, the authors investigated the role of ICa-L in regulating mitochondrial function in αMHC403/+ mice. Whole-cell patch clamp studies showed that ICa-L current inactivation kinetics were significantly increased in αMHC403/+ cardiac myocytes, but that current density and channel expression were similar to wild-type cardiac myocytes. Activation of ICa-L caused a significantly greater increase in mitochondrial membrane potential and metabolic activity in αMHC403/+ . These increases were attenuated with ICa-L antagonists and following F-actin or ß-tubulin depolymerization. The authors observed increased levels of fibroblast growth factor-21 in αMHC403/+ mice, and altered mitochondrial DNA copy number consistent with altered mitochondrial activity and the development of cardiomyopathy. These studies suggest that the Arg403Gln mutation leads to altered functional communication between ICa-L and mitochondria that is associated with increased metabolic activity, which may contribute to the development of cardiomyopathy. ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity.

Nanoparticle-mediated dual delivery of an antioxidant and a peptide against the L-Type Ca2+ channel enables simultaneous reduction of cardiac ischemia-reperfusion injury.

Increased reactive oxygen species (ROS) production and elevated intracellular Ca(2+) following cardiac ischemia-reperfusion injury are key mediators of cell death and the development of cardiac hypertrophy. The L-type Ca(2+) channel is the main route for calcium influx in cardiac myocytes. Activation of the L-type Ca(2+) channel leads to a further increase in mitochondrial ROS production and metabolism. We have previously shown that the application of a peptide derived against the alpha-interacting domain of the L-type Ca(2+) channel (AID) decreases myocardial injury post reperfusion. Herein, we examine the efficacy of simultaneous delivery of the AID peptide in combination with the potent antioxidants curcumin or resveratrol using multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles. We highlight that drug loading and dissolution are important parameters that have to be taken into account when designing novel combinatorial therapies following cardiac ischemia-reperfusion injury. In the case of resveratrol low loading capacity and fast release rates hinder its applicability as an effective candidate for simultaneous therapy. However, in the case of curcumin, high loading capacity and sustained release rates enable its effective simultaneous delivery in combination with the AID peptide. Simultaneous delivery of the AID peptide with curcumin allowed for effective attenuation of the L-type Ca(2+) channel-activated increases in superoxide (assessed as changes in DHE fluorescence; Empty NP = 53.1 ± 7.6%; NP-C-AID = 7.32 ± 3.57%) and mitochondrial membrane potential (assessed as changes in JC-1 fluorescence; Empty NP = 19.8 ± 2.8%; NP-C-AID=13.05 ± 1.78%). We demonstrate in isolated rat hearts exposed to ischemia followed by reperfusion, that curcumin and the AID peptide in combination effectively reduce muscle damage, decrease oxidative stress and superoxide production in cardiac myocytes.

Experimental Traumatic Brain Injury Results in Long-Term Recovery of Functional Responsiveness in Sensory Cortex but Persisting Structural Changes and Sensorimotor, Cognitive, and Emotional Deficits.

Traumatic brain injury (TBI) is a leading cause of death worldwide. In recent studies, we have shown that experimental TBI caused an immediate (24-h post) suppression of neuronal processing, especially in supragranular cortical layers. We now examine the long-term effects of experimental TBI on the sensory cortex and how these changes may contribute to a range of TBI morbidities. Adult male Sprague-Dawley rats received either a moderate lateral fluid percussion injury (n=14) or a sham surgery (n=12) and 12 weeks of recovery before behavioral assessment, magnetic resonance imaging, and electrophysiological recordings from the barrel cortex. TBI rats demonstrated sensorimotor deficits, cognitive impairments, and anxiety-like behavior, and this was associated with significant atrophy of the barrel cortex and other brain structures. Extracellular recordings from ipsilateral barrel cortex revealed normal neuronal responsiveness and diffusion tensor MRI showed increased fractional anisotropy, axial diffusivity, and tract density within this region. These findings suggest that long-term recovery of neuronal responsiveness is owing to structural reorganization within this region. Therefore, it is likely that long-term structural and functional changes within sensory cortex post-TBI may allow for recovery of neuronal responsiveness, but that this recovery does not remediate all behavioral deficits.

Modulation of synaptic vesicle exocytosis in muscle-dependent long-term depression at the amphibian neuromuscular junction.

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.

The acute phase of mild traumatic brain injury is characterized by a distance-dependent neuronal hypoactivity.

The consequences of mild traumatic brain injury (TBI) on neuronal functionality are only now being elucidated. We have now examined the changes in sensory encoding in the whisker-recipient barrel cortex and the brain tissue damage in the acute phase (24 h) after induction of TBI (n=9), with sham controls receiving surgery only (n=5). Injury was induced using the lateral fluid percussion injury method, which causes a mixture of focal and diffuse brain injury. Both population and single cell neuronal responses evoked by both simple and complex whisker stimuli revealed a suppression of activity that decreased with distance from the locus of injury both within a hemisphere and across hemispheres, with a greater extent of hypoactivity in ipsilateral barrel cortex compared with contralateral cortex. This was coupled with an increase in spontaneous output in Layer 5a, but only ipsilateral to the injury site. There was also disruption of axonal integrity in various regions in the ipsilateral but not contralateral hemisphere. These results complement our previous findings after mild diffuse-only TBI induced by the weight-drop impact acceleration method where, in the same acute post-injury phase, we found a similar depth-dependent hypoactivity in sensory cortex. This suggests a common sequelae of events in both diffuse TBI and mixed focal/diffuse TBI in the immediate post-injury period that then evolve over time to produce different long-term functional outcomes.

Postsynaptic protein synthesis is required for presynaptic enhancement in persistent forms of long-term potentiation.

Long-term potentiation (LTP) in the hippocampus is a fundamental process underlying learning and memory in the brain. At CA3-CA1 synapses, three discrete forms of LTP (LTP1, 2, and 3) have been differentiated on the basis of their persistence, maintenance mechanisms, Ca(2+) signaling pathways, expression loci, and electrophysiological requirements. We previously showed that LTP2 and LTP3 involve a presynaptic expression component that is established in a translation-dependent manner. Here we investigate the locus of translation required for presynaptic expression. Neurotransmitter release rate was estimated via FM 1-43 destaining from CA3 terminals in hippocampal slices from male Wistar rats (6-8 weeks). Destaining was measured at sites making putative contact with CA1 dendritic processes in stratum radiatum that had been filled with a membrane impermeable translation inhibitor and a fluorescent indicator. Our results suggest that inhibition of postsynaptic translation eliminates the enhanced release ordinarily observed at 160 min post-LTP induction, and that this effect is limited to sites closely apposed to the filled postsynaptic cell. We conclude that postsynaptic translation is required for the presynaptic component of LTP2 and LTP3 expression. These data considerably strengthen the mechanistic separation of LTP1, 2, and 3 and provide evidence for an expanded repertoire of communication between synaptic elements.