Widespread formation of double-stranded RNAs in testis.

The testis transcriptome is highly complex and includes RNAs that potentially hybridize to form double-stranded RNA (dsRNA). We isolated dsRNA using the monoclonal J2 antibody and deep-sequenced the enriched samples from testes of juvenile Dicer1 knockout mice, age-matched controls, and adult animals. Comparison of our data set with recently published data from mouse liver revealed that the dsRNA transcriptome in testis is markedly different from liver: In testis, dsRNA-forming transcripts derive from mRNAs including promoters and immediate downstream regions, whereas in somatic cells they originate more often from introns and intergenic transcription. The genes that generate dsRNA are significantly expressed in isolated male germ cells with particular enrichment in pachytene spermatocytes. dsRNA formation is lower on the sex (X and Y) chromosomes. The dsRNA transcriptome is significantly less complex in juvenile mice as compared to adult controls and, possibly as a consequence, the knockout of Dicer1 has only a minor effect on the total number of transcript peaks associated with dsRNA. The comparison between dsRNA-associated genes in testis and liver with a reported set of genes that produce endogenous siRNAs reveals a significant overlap in testis but not in liver. Testis dsRNAs also significantly associate with natural antisense genes-again, this feature is not observed in liver. These findings point to a testis-specific mechanism involving natural antisense transcripts and the formation of dsRNAs that feed into the RNA interference pathway, possibly to mitigate the mutagenic impacts of recombination and transposon mobilization.

A small switch in perspective: Comparing weight loss by nutrient balance versus caloric balance.

The establishment of a Caloric balance has been classically discussed as the means to induce weight loss. Recently, the idea of nutrient balance as opposed to Caloric balance has emerged as a better means to induce weight loss. This investigation compared differences in weight loss between a diet based on a nutrient balanced diet compared to a Caloric balance diet. 53 (27M/26F) active overfat individuals (30.7+/- 7.1 years) were randomly (matched for age, gender, training history) assigned within an 8-week intervention to follow either a self-selected diet (control) or a diet based on following a Caloric balance (%Cal/day) or a nutrient balance (g/kg/day) in conjunction with a periodized exercise regimen to determine effectiveness for each diet to induce weight loss. Nutrient balance group had significantly different changes (p < 0.05) in fat-free mass (2.26 (2.02, 2.49) kg versus 0.42 (-0.40, 1.24) kg) and fat mass (-5.96 (-5.34, -6.58) kg versus -4.08 (-3.92, -5.92) kg) relative to the Caloric balance group and was more effective at meeting nutritional requirements for protein (ES = 0.65 (0.48, 0.85)) and lipids (ES = 0.24 (-0.09, 0.98)) than the Caloric balance group. Nutrient balance was subjectively scored as easier to follow and more likely to be self-selected. Using a nutrient balance diet may be more effective at inducing beneficial body compositional changes and shows being a more self-selected dietary method when compared to a Caloric balance diet. Therefore, it may be a better choice for advice when offering treatments to those who are attempting to lose weight or maintain weight loss.

Modelling mood updating: a proof of principle study.

BACKGROUND: Recent developments in computational psychiatry have led to the hypothesis that mood represents an expectation (prior belief) on the likely interoceptive consequences of action (i.e. emotion). This stems from ideas about how the brain navigates its external world by minimising an upper bound on surprisal (free energy) of sensory information and echoes developments in other perceptual domains. AIMS: In this paper we aim to present a simple partial observable Markov decision process that models mood updating in response to stressful or non-stressful environmental fluctuations while seeking to minimise surprisal in relation to prior beliefs about the likely interoceptive signals experienced with specific actions (attenuating or amplifying stress and pleasure signals). METHOD: We examine how, by altering these prior beliefs we can model mood updating in depression, mania and anxiety. RESULTS: We discuss how these models provide a computational account of mood and its related psychopathology and relate it to previous research in reward processing. CONCLUSIONS: Models such as this can provide hypotheses for experimental work and also open up the potential modelling of predicted disease trajectories in individual patients.

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. METHODS: A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and ex vivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. RESULTS: MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. CONCLUSIONS: Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.

Comparing effectiveness of fat burners and thermogenic supplements to diet and exercise for weight loss and cardiometabolic health: Systematic review and meta-analysis.

BACKGROUND: Those who are overfat face an onslaught of advice for losing weight, including using dietary supplements that purport to have fat burning capabilities to achieve a reduced body mass, fat mass and improvement in cardiometabolic health in combination with exercise or diet and exercise regimens. AIM: To examine long-term effectiveness of supplements for both weight loss and improvements in cardiometabolic health for these individuals. METHODS: A PRISMA methods of systematic review was conducted from August 2018 through January 2019 using Medline, PubChem, PubMed, EBOSCO CINHAL and SPORTDiscus, and Google Scholar yielding 23,441 returns of which 21 studies (duration greater than 8 weeks with participant populations of BMI greater than 24.9) were included for meta-analysis. Meta-analysis examined pooled effect size and 95% confidence interval for: body mass, fat mass, fat-free mass, total cholesterol, high-density lipoproteins, low-density lipoproteins, resting metabolic rate. Intra-study effect sizes were compared with previously reported results for diet or diet and exercise in a 2x2 chi-square analysis for the number of studies that induced effects greater than or less than the effect size. RESULTS: There is a general trend to show effectiveness (effect size greater than 0.00) for obtaining beneficial changes from use of thermogenic dietary supplements, yet the 95% confidence interval for effect size crossed 0.00 (indicating no benefit). Chi-square comparison to exercise, or combination of diet and exercise, indicates that responses induced from weight-loss supplements were less effective than what is obtained from utilizing exercise, or diet and exercise, without additional weight-loss supplements. CONCLUSION: There appears to be limited benefit that may be derived from the inclusion of thermogenic dietary supplements to reduce body mass and improve cardiometabolic health for individuals who are overfat.

p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.

Despite the high and preferential expression of p38γ MAPK in the myocardium, little is known about its function in the heart. The aim of the current study was to elucidate the physiologic and biochemical roles of p38γ in the heart. Expression and subcellular localization of p38 isoforms was determined in mouse hearts. Comparisons of the cardiac function and structure of wild-type and p38γ knockout (KO) mice at baseline and after abdominal aortic banding demonstrated that KO mice developed less ventricular hypertrophy and that contractile function is better preserved. To identify potential substrates of p38γ, we generated an analog-sensitive mutant to affinity tag endogenous myocardial proteins. Among other proteins, this technique identified calpastatin as a direct p38γ substrate. Moreover, phosphorylation of calpastatin by p38γ impaired its ability to inhibit the protease, calpain. We have identified p38γ as an important determinant of the progression of pathologic cardiac hypertrophy after aortic banding in mice. In addition, we have identified calpastatin, among other substrates, as a novel direct target of p38γ that may contribute to the protection observed in p38γKO mice.-Loonat, A. A., Martin, E. D., Sarafraz-Shekary, N., Tilgner, K., Hertz, N. T., Levin, R., Shokat, K. M., Burlingame, A. L., Arabacilar, P., Uddin, S., Thomas, M., Marber, M. S., Clark, J. E. p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.

Dynamic heart rate response to multi-day unsupported ultra-endurance cycle racing: a case report.

NEW FINDINGS: What is the main observation in this case? Ultra-endurance cycle racing is known to lead to suppressed heart rates as a product of time spent racing. This case report identifies a racer who experienced this phenomenon initially, but then uniquely experienced an overall increase in heart rate late in the race. What insight does it reveal? In this case, unique chronotropic disturbances to heart rate occurred as a result of the many extreme demands of ultra-endurance racing. Work should now focus on identifying the frequency of this response in other racers and whether the main causes are physiological, environmental or genetic in nature. ABSTRACT: Participation in ultra-endurance cycling events, such as the Transcontinental Race, is increasing. These extremely demanding races provide a unique opportunity for field observation of the limits of human endurance physiology and, importantly, when these limits might be exceeded and cross over into pathology. The heart is of special interest in this field, and previous data suggest that 'reverse drift' of heart rate occurs as a product of time and load in races of 24-48 h, whereas transient structural abnormalities have been observed upon completion of running ultramarathons. Here, we report a unique case of a male cyclist racing in the Transcontinental Race over an extended period of 14 days characterized by extreme workloads and a low quantity and quality of sleep. The heart rate response was dynamic over the course of the race and defined by a U-shaped quadratic relationship. A larger scale study is required to determine the relevance of this information to the ultra-endurance cycling community.

A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy, and Heart Failure.

BACKGROUND: Research into the therapeutic potential of α-calcitonin gene-related peptide (α-CGRP) has been limited because of its peptide nature and short half-life. Here, we evaluate whether a novel potent and long-lasting (t½ ≥7 hours) acylated α-CGRP analogue (αAnalogue) could alleviate and reverse cardiovascular disease in 2 distinct murine models of hypertension and heart failure in vivo. METHODS: The ability of the αAnalogue to act selectively via the CGRP pathway was shown in skin by using a CGRP receptor antagonist. The effect of the αAnalogue on angiotensin II-induced hypertension was investigated over 14 days. Blood pressure was measured by radiotelemetry. The ability of the αAnalogue to modulate heart failure was studied in an abdominal aortic constriction model of murine cardiac hypertrophy and heart failure over 5 weeks. Extensive ex vivo analysis was performed via RNA analysis, Western blot, and histology. RESULTS: The angiotensin II-induced hypertension was attenuated by cotreatment with the αAnalogue (50 nmol·kg-1·d-1, SC, at a dose selected for lack of long-term hypotensive effects at baseline). The αAnalogue protected against vascular, renal, and cardiac dysfunction, characterized by reduced hypertrophy and biomarkers of fibrosis, remodeling, inflammation, and oxidative stress. In a separate study, the αAnalogue reversed angiotensin II-induced hypertension and associated vascular and cardiac damage. The αAnalogue was effective over 5 weeks in a murine model of cardiac hypertrophy and heart failure. It preserved heart function, assessed by echocardiography, while protecting against adverse cardiac remodeling and apoptosis. Moreover, treatment with the αAnalogue was well tolerated with neither signs of desensitization nor behavioral changes. CONCLUSIONS: These findings, in 2 distinct models, provide the first evidence for the therapeutic potential of a stabilized αAnalogue, by mediating (1) antihypertensive effects, (2) attenuating cardiac remodeling, and (3) increasing angiogenesis and cell survival to protect against and limit damage associated with the progression of cardiovascular diseases. This indicates the therapeutic potential of the CGRP pathway and the possibility that this injectable CGRP analogue may be effective in cardiac disease.

What is mood? A computational perspective.

The neurobiological understanding of mood, and by extension mood disorders, remains elusive despite decades of research implicating several neuromodulator systems. This review considers a new approach based on existing theories of functional brain organisation. The free energy principle (a.k.a. active inference), and its instantiation in the Bayesian brain, offers a complete and simple formulation of mood. It has been proposed that emotions reflect the precision of - or certainty about - the predicted sensorimotor/interoceptive consequences of action. By extending this reasoning, in a hierarchical setting, we suggest mood states act as (hyper) priors over uncertainty (i.e. emotions). Here, we consider the same computational pathology in the proprioceptive and interoceptive (behavioural and autonomic) domain in order to furnish an explanation for mood disorders. This formulation reconciles several strands of research at multiple levels of enquiry.

The aetiopathogenesis of fatigue: unpredictable, complex and persistent.

BACKGROUND: Chronic fatigue syndrome is a common condition characterized by severe fatigue with post-exertional malaise, impaired cognitive ability, poor sleep quality, muscle pain, multi-joint pain, tender lymph nodes, sore throat or headache. Its defining symptom, fatigue is common to several diseases. AREAS OF AGREEMENT: Research has established a broad picture of impairment across autonomic, endocrine and inflammatory systems though progress seems to have reached an impasse. AREAS OF CONTROVERSY: The absence of a clear consensus view of the pathophysiology of fatigue suggests the need to switch from a focus on abnormalities in one system to an experimental and clinical approach which integrates findings across multiple systems and their constituent parts and to consider multiple environmental factors. GROWING POINTS: We discuss this with reference to three key factors, non-determinism, non-reductionism and self-organization and suggest that an approach based on these principles may afford a coherent explanatory framework for much of the observed phenomena in fatigue and offers promising avenues for future research. AREAS TIMELY FOR DEVELOPING RESEARCH: By adopting this approach, the field can examine issues regarding aetiopathogenesis and treatment, with relevance for future research and clinical practice.

A simple method for optimising transformation of non-parametric data: an illustration by reference to cortisol assays.

Neuroendocrine data are typically positively skewed and rarely conform to the expectations of a Gaussian distribution. This can be a problem when attempting to analyse results within the framework of the general linear model, which relies on assumptions that residuals in the data are normally distributed. One frequently used method for handling violations of this assumption is to transform variables to bring residuals into closer alignment with assumptions (as residuals are not directly manipulated). This is often attempted through ad hoc traditional transformations such as square root, log and inverse. However, Box and Cox (Box & Cox, ) observed that these are all special cases of power transformations and proposed a more flexible method of transformation for researchers to optimise alignment with assumptions. The goal of this paper is to demonstrate the benefits of the infinitely flexible Box-Cox transformation on neuroendocrine data using syntax in spss. When applied to positively skewed data typical of neuroendocrine data, the majority (~2/3) of cases were brought into strict alignment with Gaussian distribution (i.e. a non-significant Shapiro-Wilks test). Those unable to meet this challenge showed substantial improvement in distributional properties. The biggest challenge was distributions with a high ratio of kurtosis to skewness. We discuss how these cases might be handled, and we highlight some of the broader issues associated with transformation. Copyright © 2016 John Wiley & Sons, Ltd.

Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury.

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2-deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti-MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.

Correction: Ruchaya et al. Transplantation of Skeletal Muscle-Derived Sca-1+/PW1+/Pax7- Interstitial Cells (PICs) Improves Cardiac Function and Attenuates Remodeling in Mice Subjected to Myocardial Infarction. Cells2022, 11, 4050.

In the original publication [...].

Transient Receptor Potential Canonical 5 (TRPC5): Regulation of Heart Rate and Protection against Pathological Cardiac Hypertrophy.

TRPC5 is a non-selective cation channel that is expressed in cardiomyocytes, but there is a lack of knowledge of its (patho)physiological role in vivo. Here, we examine the role of TRPC5 on cardiac function under basal conditions and during cardiac hypertrophy. Cardiovascular parameters were assessed in wild-type (WT) and global TRPC5 knockout (KO) mice. Despite no difference in blood pressure or activity, heart rate was significantly reduced in TRPC5 KO mice. Echocardiography imaging revealed an increase in stroke volume, but cardiac contractility was unaffected. The reduced heart rate persisted in isolated TRPC5 KO hearts, suggesting changes in basal cardiac pacing. Heart rate was further investigated by evaluating the reflex change following drug-induced pressure changes. The reflex bradycardic response following phenylephrine was greater in TRPC5 KO mice but the tachycardic response to SNP was unchanged, indicating an enhancement in the parasympathetic control of the heart rate. Moreover, the reduction in heart rate to carbachol was greater in isolated TRPC5 KO hearts. To evaluate the role of TRPC5 in cardiac pathology, mice were subjected to abdominal aortic banding (AAB). An exaggerated cardiac hypertrophy response to AAB was observed in TRPC5 KO mice, with an increased expression of hypertrophy markers, fibrosis, reactive oxygen species, and angiogenesis. This study provides novel evidence for a direct effect of TRPC5 on cardiac function. We propose that (1) TRPC5 is required for maintaining heart rate by regulating basal cardiac pacing and in response to pressure lowering, and (2) TRPC5 protects against pathological cardiac hypertrophy.

Advancements in pressure-volume catheter technology - stress remodelling after infarction.

Microconductance catheters have been successfully applied to measure left ventricular (LV) function in the mouse to assess cardiac or pharmacological interventions for a number of years. New complex admittance methods produce an estimate of the parallel admittance of cardiac muscle that can be used to correct the measurement in real time. This contrasts with existing conductance technologies that require in vivo calibration using a bolus of hypertonic saline. Here, we report the application of this emerging technology in the context of myocardial infarction and LV remodelling. Using a combination of high-resolution ultrasound and LV conductance catheters, we compared measures of LV function using an admittance system and a traditional conductance-derived pressure-volume (PV) system. We subjected C57BL/6 mice to focal myocardial ischaemia-reperfusion by transient ligation of the left anterior descending coronary artery and assessed cardiac function with different systems to determine the reliability and accuracy of these methods to distinguish between normal and dysfunctional ventricle. We demonstrate that the admittance PV system, in our hands, provides a straightforward solution for assessing LV function in mice. Using this technique in combination with other established methods, we measured LV dysfunction following coronary artery occlusion and reperfusion, which can be ameliorated using a known preconditioning agent (CORM-3), and found that functional read-outs are representative of other methods. We have found that, especially in diseased tissue, LV pressure-volume loops derived from complex admittance provide a reproducible and reliable method of determining LV function without the need for technically challenging calibration. Our data suggest that admittance records accurate/physiological LV cavity volumes when compared with other invasive methods in the same animal. This emerging technology is both effective and reproducible for measuring LV function and dysfunction in the mouse, without the need for complicated interventions to calibrate the measurements or training in a new technology. This may mark the way towards a fast and accurate assessment of murine cardiac function in normal animals and disease models.

The impact of bias on developing healthy lifestyles, understanding if personal perspectives impact modeling and recommendations of diet and exercise by educators to their students.

Purpose: Educators have an ability to imprint healthy behavior in children. Yet, little is known about how a bias by educators might impact imprinting on students. Therefore, we examined if educators' bias in opinions about diet and exercise influence the manner they are discussed with students. Methods: 340 (144 F/196 M) educators from over 14 states (USA) provided responses regarding: personal opinions about and history of following diets or using exercise regimens; perspective on commonly held beliefs regarding diet, exercise, body image and morphology; and who should provide recommendations. Responses were tabulated for average and percentage with subsequent analysis by Pearson correlations or keyword frequencies of responses. Results: Almost all (97%) understand social pressures related to body image and need to portray healthy behaviors to students. Bias was evident based on history of recommending or discouraging a specific diet (r = 0.77) or a dietary supplement (r = 0.66), recommending exercise they used (r = 0.89) or discouraging ones not used (r = 0.65). Most (85%) understand that social and mass media are not reliable sources, yet, relied on the same sources for information that reinforced their opinions. Conclusion: Findings indicate (1) portrayal of healthy behaviors to students exist but expressed opinion that families have a greater influence than educators on healthy lifestyles, (2) there appears to be an unawareness of personal bias or expression of implicit bias toward behaviors projected to students, and (3) health/physical education and life science teachers may be able to act as a source of unbiased information to provide resources to a school site to aid in developing healthy lifestyles.

Transplantation of Skeletal Muscle-Derived Sca-1+/PW1+/Pax7- Interstitial Cells (PICs) Improves Cardiac Function and Attenuates Remodeling in Mice Subjected to Myocardial Infarction.

We have previously shown that skeletal muscle-derived Sca-1+/PW1+/Pax7- interstitial cells (PICs) are multi-potent and enhance endogenous repair and regeneration. Here, we investigated the regenerative potential of PICs following intramyocardial transplantation in mice subjected to an acute myocardial infarction (MI). MI was induced through the ligation of the left anterior descending coronary artery in 8-week old male C57BL/6 mice. 5 × 105 eGFP-labelled PICs (MI + PICs; n = 7) or PBS (MI-PBS; n = 7) were injected intramyocardially into the border zone. Sham mice (n = 8) were not subjected to MI, or the transplantation of PICs or PBS. BrdU was administered via osmotic mini-pump for 14 days. Echocardiography was performed prior to surgery (baseline), and 1-, 3- and 6-weeks post-MI and PICs transplantation. Mice were sacrificed at 6 weeks post-MI + PICs transplantation, and heart sections were analysed for fibrosis, hypertrophy, engraftment, proliferation, and differentiation of PICs. A significant (p < 0.05) improvement in ejection fraction (EF) and fractional shortening was observed in the MI-PICs group, compared to MI + PBS group at 6-weeks post MI + PICs transplantation. Infarct size/fibrosis of the left ventricle significantly (p < 0.05) decreased in the MI-PICs group (14.0 ± 2.5%), compared to the MI-PBS group (32.8 ± 2.2%). Cardiomyocyte hypertrophy in the border zone significantly (p < 0.05) decreased in the MI-PICs group compared to the MI-PBS group (330.0 ± 28.5 µM2 vs. 543.5 ± 26.6 µm2), as did cardiomyocyte apoptosis (0.6 ± 0.9% MI-PICs vs. 2.8 ± 0.8% MI-PBS). The number of BrdU+ cardiomyocytes was significantly (p < 0.05) increased in the infarct/border zone of the MI-PICs group (7.0 ± 3.3%), compared to the MI-PBS group (1.7 ± 0.5%). The proliferation index (total BrdU+ cells) was significantly increased in the MI-PICs group compared to the MI-PBS group (27.0 ± 3.4% vs. 7.6 ± 1.0%). PICs expressed and secreted pro-survival and reparative growth factors, supporting a paracrine effect of PICs during recovery/remodeling. Skeletal muscle-derived PICs show significant reparative potential, attenuating cardiac remodelling following transplantation into the infarcted myocardium. PICs can be easily sourced from skeletal muscle and therefore show promise as a potential cell candidate for supporting the reparative and regenerative effects of cell therapies.

A chemical genetic approach reveals that p38alpha MAPK activation by diphosphorylation aggravates myocardial infarction and is prevented by the direct binding of SB203580.

The use of nonselective pharmacological inhibitors has resulted in controversy regarding the mechanism and consequences of p38 activation during myocardial infarction. Classic p38 inhibitors such as SB203580 rely on a critical "gatekeeper" threonine residue for binding. We addressed these controversies by using mice in which the p38alpha alleles were targeted to cause substitution of the gatekeeper residue and resistance to inhibition. In homozygous drug-resistant compared with wild-type hearts, SB203580 failed to inhibit the activating phosphorylation of p38 or to reduce the infarction caused by myocardial ischemia. However, BIRB796, a p38 inhibitor not reliant on the gatekeeper for binding, similarly reduced p38-activating phosphorylation and infarction in both wild-type and knock-in mice, thereby excluding a nonspecific inhibitor-dependent phenotype resulting from the targeting strategy. Furthermore, the activation during myocardial ischemia involved phosphorylation of both the threonine and tyrosine residues in the activation loop of p38 despite the phosphorylation of the threonine alone being sufficient to create the epitope for dual phosphospecific antibody binding. Finally, SB203580 failed to reduce infarction in heterozygous drug-resistant hearts, suggesting that near complete inhibition of p38alpha kinase activity is necessary to elicit protection. These results indicate that, during myocardial ischemia, p38alpha (i) is the dominant-active p38 isoform, (ii) contributes to infarction, (iii) is responsible for the cardioprotective effect of SB203580, and (iv) is activated by a mechanism consistent with autodiphosphorylation despite this necessitating the phosphorylation of a tyrosine residue by an archetypal serine/threonine kinase.

Myocardial stress remodelling after regional infarction is independent of glycogen synthase kinase-3 inactivation.

Phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3) is observed in the failing heart induced by chronic pharmacological stress and aortic banding. Constitutive kinase activity attenuates pathological remodelling, suggesting an obligatory role in stress signalling. However, this has been challenged by recent data whereby conditional GSK-3ß deletion has been shown to protect against post-infarct remodelling. Here, we set out to determine the chronic remodelling response to infarction in hearts of GSK-3α/ß(Ala21/9) knockin (KI) mice encoding constitutively active GSK-3 isoforms. At 4 weeks after infarction there were significant increases in normalised heart weight and left ventricular (LV) muscle volume compared to sham in both KI and wild type animals. This was associated with an increase in LV cavity dimensions and remote LV wall thickness. Hypertrophy in both genotypes resulted in marked contractile impairment on both invasive and non-invasive interrogation. Increased phosphorylation of GSK-3ß, but not GSK-3α, was demonstrated at 1 week after infarction and remained elevated at 4 weeks compared to sham-treated hearts. In conclusion, GSK-3ß phosphorylation and inactivation occurs with, but is not an obligatory signalling event in, chronic post-infarct remodelling in the mouse heart. This highlights the heterogeneity of pathological hypertrophy and the divergent role of GSK-3 signalling in chronic myocardial stress.

The activation of p38 alpha, and not p38 beta, mitogen-activated protein kinase is required for ischemic preconditioning.

Numerous studies show that pharmacological inhibition of p38 mitogen-activated protein kinases (p38s) before lethal ischemia prevents conditioning. However, these inhibitors have off-target effects and do not discriminate between the alpha and beta isoforms; the activation of which is thought to have diverse and perhaps opposing actions with p38 alpha aggravating, and p38 beta reducing, myocardial injury. We adopted a chemical genetic approach using mice in which either the p38 alpha (DR alpha) or p38 beta (DR beta) alleles were targeted to substitute the "gatekeeper" threonine residue for methionine, thereby preventing the binding of a pharmacological inhibitor, SB203580. Isolated, perfused wild-type (WT), DR alpha and DR beta mouse hearts underwent ischemic preconditioning with 4 cycles of 4 min ischemia/6 min reperfusion, with or without SB203580 (10 microM), followed by 30 min of global ischemia and 120 min of reperfusion. In WT and DR beta hearts, SB203580 completely abolished the reduction in myocardial infarction seen with preconditioning and also the phosphorylation of downstream substrates of p38. These effects of SB203580 were not seen in DR alpha hearts. Furthermore ischemic preconditioning occurred unaltered in p38 beta null hearts. Contrary to expectation the activation of p38 alpha, and not p38 beta, is necessary for ischemic preconditioning. Since p38 alpha is also the isoform that leads to lethal myocardial injury, it is unlikely that targeted therapeutic strategies to achieve isoform-selective inhibition will only prevent the harmful consequences of activation.