Metformin: Experimental and Clinical Evidence for a Potential Role in Emphysema Treatment.

Rationale: Cigarette smoke (CS) inhalation triggers oxidative stress and inflammation, leading to accelerated lung aging, apoptosis, and emphysema, as well as systemic pathologies. Metformin is beneficial for protecting against aging-related diseases. Objectives: We sought to investigate whether metformin may ameliorate CS-induced pathologies of emphysematous chronic obstructive pulmonary disease (COPD). Methods: Mice were exposed chronically to CS and fed metformin-enriched chow for the second half of exposure. Lung, kidney, and muscle pathologies, lung proteostasis, endoplasmic reticulum (ER) stress, mitochondrial function, and mediators of metformin effects in vivo and/or in vitro were studied. We evaluated the association of metformin use with indices of emphysema progression over 5 years of follow-up among the COPDGene (Genetic Epidemiology of COPD) study participants. The association of metformin use with the percentage of emphysema and adjusted lung density was estimated by using a linear mixed model. Measurements and Main Results: Metformin protected against CS-induced pulmonary inflammation and airspace enlargement; small airway remodeling, glomerular shrinkage, oxidative stress, apoptosis, telomere damage, aging, dysmetabolism in vivo and in vitro; and ER stress. The AMPK (AMP-activated protein kinase) pathway was central to metformin's protective action. Within COPDGene, participants receiving metformin compared with those not receiving it had a slower progression of emphysema (-0.92%; 95% confidence interval [CI], -1.7% to -0.14%; P = 0.02) and a slower adjusted lung density decrease (2.2 g/L; 95% CI, 0.43 to 4.0 g/L; P = 0.01). Conclusions: Metformin protected against CS-induced lung, renal, and muscle injury; mitochondrial dysfunction; and unfolded protein responses and ER stress in mice. In humans, metformin use was associated with lesser emphysema progression over time. Our results provide a rationale for clinical trials testing the efficacy of metformin in limiting emphysema progression and its systemic consequences.

Neurofilament light: a possible prognostic biomarker for treatment of vascular contributions to cognitive impairment and dementia.

BACKGROUND: Decreased cerebral blood flow and systemic inflammation during heart failure (HF) increase the risk for vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer disease-related dementias (ADRD). We previously demonstrated that PNA5, a novel glycosylated angiotensin 1-7 (Ang-(1-7)) Mas receptor (MasR) agonist peptide, is an effective therapy to rescue cognitive impairment in our preclinical model of VCID. Neurofilament light (NfL) protein concentration is correlated with cognitive impairment and elevated in neurodegenerative diseases, hypoxic brain injury, and cardiac disease. The goal of the present study was to determine (1) if treatment with Ang-(1-7)/MasR agonists can rescue cognitive impairment and decrease VCID-induced increases in NfL levels as compared to HF-saline treated mice and, (2) if NfL levels correlate with measures of cognitive function and brain cytokines in our VCID model. METHODS: VCID was induced in C57BL/6 male mice via myocardial infarction (MI). At 5 weeks post-MI, mice were treated with daily subcutaneous injections for 24 days, 5 weeks after MI, with PNA5 or angiotensin 1-7 (500 microg/kg/day or 50 microg/kg/day) or saline (n = 15/group). Following the 24-day treatment protocol, cognitive function was assessed using the Novel Object Recognition (NOR) test. Cardiac function was measured by echocardiography and plasma concentrations of NfL were quantified using a Quanterix Simoa assay. Brain and circulating cytokine levels were determined with a MILLIPLEX MAP Mouse High Sensitivity Multiplex Immunoassay. Treatment groups were compared via ANOVA, significance was set at p < 0.05. RESULTS: Treatment with Ang-(1-7)/MasR agonists reversed VCID-induced cognitive impairment and significantly decreased NfL levels in our mouse model of VCID as compared to HF-saline treated mice. Further, NfL levels were significantly negatively correlated with cognitive scores and the concentrations of multiple pleiotropic cytokines in the brain. CONCLUSIONS: These data show that treatment with Ang-(1-7)/MasR agonists rescues cognitive impairment and decreases plasma NfL relative to HF-saline-treated animals in our VCID mouse model. Further, levels of NfL are significantly negatively correlated with cognitive function and with several brain cytokine concentrations. Based on these preclinical findings, we propose that circulating NfL might be a candidate for a prognostic biomarker for VCID and may also serve as a pharmacodynamic/response biomarker for therapeutic target engagement.

Lactobacillus reuteri attenuates cardiac injury without lowering cholesterol in low-density lipoprotein receptor-deficient mice fed standard chow.

Disruption of the normal gut microbiome (dysbiosis) is implicated in the progression and severity of myriad disorders, including hypercholesterolemia and cardiovascular disease. Probiotics attenuate and reverse gut dysbiosis to improve cardiovascular risk factors like hypertension and hypercholesterolemia. Lactobacillus reuteri is a well-studied lactic acid-producing probiotic with known cholesterol-lowering properties and anti-inflammatory effects. In the present study, we hypothesized that L. reuteri delivered to hypercholesterolemic low-density lipoprotein receptor knockout (LDLr KO) mice will reduce cholesterol levels and minimize cardiac injury from an ischemic insult. L. reuteri [1 × 109 or 50 × 106 colony-forming units (CFU)/day] was administered by oral gavage to wild-type mice and LDLr KO for up to 6 wk followed by an ischemia-reperfusion (I/R) protocol. After 4 wk of gavage, total serum cholesterol in wild-type mice receiving saline was 113.5 ± 5.6 mg/dL compared with 113.3 ± 6.8 and 101.9 ± 7.5 mg/dL in mice receiving 1 × 109 or 50 × 106 CFU/day, respectively. Over the same time frame, administration of L. reuteri at 1 × 109 or 50 × 106 CFU/day did not lower total serum cholesterol (283.0 ± 11.1, 263.3 ± 5.0, and 253.1 ± 7.0 mg/dL; saline, 1 × 109 or 50 × 106 CFU/day, respectively) in LDLr KO mice. Despite no impact on total serum cholesterol, L. reuteri administration significantly attenuated cardiac injury following I/R, as evidenced by smaller infarct sizes compared with controls in both wild-type and LDLr KO groups. In conclusion, daily L. reuteri significantly protected against cardiac injury without lowering cholesterol levels, suggesting anti-inflammatory properties of L. reuteri uncoupled from improvements in serum cholesterol.NEW & NOTEWORTHY We demonstrated that daily delivery of Lactobacillus reuteri to wild-type and hypercholesterolemic lipoprotein receptor knockout mice attenuated cardiac injury following ischemia-reperfusion without lowering total serum cholesterol in the short term. In addition, we validated protection against cardiac injury using histology and immunohistochemistry techniques. L. reuteri offers promise as a probiotic to mitigate ischemic cardiac injury.

Using 4-vinylcyclohexene diepoxide as a model of menopause for cardiovascular disease.

There is a sharp rise in cardiovascular disease (CVD) risk and progression with the onset of menopause. The 4-vinylcyclohexene diepoxide (VCD) model of menopause recapitulates the natural, physiological transition through perimenopause to menopause. We hypothesized that menopausal female mice were more susceptible to CVD than pre- or perimenopausal females. Female mice were treated with VCD or vehicle for 20 consecutive days. Premenopausal, perimenopausal, and menopausal mice were administered angiotensin II (ANG II) or subjected to ischemia-reperfusion (I/R). Menopausal females were more susceptible to pathological ANG II-induced cardiac remodeling and cardiac injury from a myocardial infarction (MI), while perimenopausal, like premenopausal, females remained protected. Specifically, ANG II significantly elevated diastolic (130.9 ± 6.0 vs. 114.7 ± 6.2 mmHg) and systolic (156.9 ± 4.8 vs. 141.7 ± 5.0 mmHg) blood pressure and normalized cardiac mass (15.9 ± 1.0 vs. 7.7 ± 1.5%) to a greater extent in menopausal females compared with controls, whereas perimenopausal females demonstrated a similar elevation of diastolic (93.7 ± 2.9 vs. 100.5 ± 4.1 mmHg) and systolic (155.9 ± 7.3 vs. 152.3 ± 6.5 mmHg) blood pressure and normalized cardiac mass (8.3 ± 2.1 vs. 7.5 ± 1.4%) compared with controls. Similarly, menopausal females demonstrated a threefold increase in fibrosis measured by Picrosirus red staining. Finally, hearts of menopausal females (41 ± 5%) showed larger infarct sizes following I/R injury than perimenopausal (18.0 ± 5.6%) and premenopausal (16.2 ± 3.3, 20.1 ± 4.8%) groups. Using the VCD model of menopause, we provide evidence that menopausal females were more susceptible to pathological cardiac remodeling. We suggest that the VCD model of menopause may be critical to better elucidate cellular and molecular mechanisms underlying the transition to CVD susceptibility in menopausal women.NEW & NOTEWORTHY Before menopause, women are protected against cardiovascular disease (CVD) compared with age-matched men; this protection is gradually lost after menopause. We present the first evidence that demonstrates menopausal females are more susceptible to pathological cardiac remodeling while perimenopausal and cycling females are not. The VCD model permits appropriate examination of how increased susceptibility to the pathological process of cardiac remodeling accelerates from pre- to perimenopause to menopause.

A Novel Angiotensin-(1-7) Glycosylated Mas Receptor Agonist for Treating Vascular Cognitive Impairment and Inflammation-Related Memory Dysfunction.

Increasing evidence indicates that decreased brain blood flow, increased reactive oxygen species (ROS) production, and proinflammatory mechanisms accelerate neurodegenerative disease progression such as that seen in vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer's disease and related dementias. There is a critical clinical need for safe and effective therapies for the treatment and prevention of cognitive impairment known to occur in patients with VCID and chronic inflammatory diseases such as heart failure (HF), hypertension, and diabetes. This study used our mouse model of VCID/HF to test our novel glycosylated angiotensin-(1-7) peptide Ang-1-6-O-Ser-Glc-NH2 (PNA5) as a therapy to treat VCID and to investigate circulating inflammatory biomarkers that may be involved. We demonstrate that PNA5 has greater brain penetration compared with the native angiotensin-(1-7) peptide. Moreover, after treatment with 1.0/mg/kg, s.c., for 21 days, PNA5 exhibits up to 10 days of sustained cognitive protective effects in our VCID/HF mice that last beyond the peptide half-life. PNA5 reversed object recognition impairment in VCID/HF mice and rescued spatial memory impairment. PNA5 activation of the Mas receptor results in a dose-dependent inhibition of ROS in human endothelial cells. Last, PNA5 treatment decreased VCID/HF-induced activation of brain microglia/macrophages and inhibited circulating tumor necrosis factor α, interleukin (IL)-7, and granulocyte cell-stimulating factor serum levels while increasing that of the anti-inflammatory cytokine IL-10. These results suggest that PNA5 is an excellent candidate and "first-in-class" therapy for treating VCID and other inflammation-related brain diseases.

Cardiac-specific knockout of Lmod2 results in a severe reduction in myofilament force production and rapid cardiac failure.

Leiomodin-2 (Lmod2) is a striated muscle-specific actin binding protein that is implicated in assembly of thin filaments. The necessity of Lmod2 in the adult mouse and role it plays in the mechanics of contraction are unknown. To answer these questions, we generated cardiac-specific conditional Lmod2 knockout mice (cKO). These mice die within a week of induction of the knockout with severe left ventricular systolic dysfunction and little change in cardiac morphology. Cardiac trabeculae isolated from cKO mice have a significant decrease in maximum force production and a blunting of myofilament length-dependent activation. Thin filaments are non-uniform and substantially reduced in length in cKO hearts, affecting the functional overlap of the thick and thin filaments. Remarkably, we also found that Lmod2 levels are directly linked to thin filament length and cardiac function in vivo, with a low amount (<20%) of Lmod2 necessary to maintain cardiac function. Thus, Lmod2 plays an essential role in maintaining proper cardiac thin filament length in adult mice, which in turn is necessary for proper generation of contractile force. Dysregulation of thin filament length in the absence of Lmod2 contributes to heart failure.

Nebulin deficiency in adult muscle causes sarcomere defects and muscle-type-dependent changes in trophicity: novel insights in nemaline myopathy.

Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods and undergo fiber-type switching toward oxidative types. Nebulin deficiency causes a large deficit in specific force, and mechanistic studies provide evidence that a reduced fraction of force-generating cross-bridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas (CSAs), worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even have hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of NEM (muscle weakness, oxidative fiber-type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.

Sex dimorphisms of crossbridge cycling kinetics in transgenic hypertrophic cardiomyopathy mice.

Familial hypertrophic cardiomyopathy (HCM) is a disease of the sarcomere and may lead to hypertrophic, dilated, restrictive, and/or arrhythmogenic cardiomyopathy, congestive heart failure, or sudden cardiac death. We hypothesized that hearts from transgenic HCM mice harboring a mutant myosin heavy chain increase the energetic cost of contraction in a sex-specific manner. To do this, we assessed Ca(2+) sensitivity of tension and crossbridge kinetics in demembranated cardiac trabeculas from male and female wild-type (WT) and HCM hearts at an early time point (2 mo of age). We found a significant effect of sex on Ca(2+) sensitivity such that male, but not female, HCM mice displayed a decrease in Ca(2+) sensitivity compared with WT counterparts. The HCM transgene and sex significantly impacted the rate of force redevelopment by a rapid release-restretch protocol and tension cost by the ATPase-tension relationship. In each of these measures, HCM male trabeculas displayed a gain-of-function when compared with WT counterparts. In addition, cardiac remodeling measured by echocardiography, histology, morphometry, and posttranslational modifications demonstrated sex- and HCM-specific effects. In conclusion, female and male HCM mice display sex dimorphic crossbridge kinetics accompanied by sex- and HCM-dependent cardiac remodeling at the morphometric, histological, and cellular level.

Liver Kinase B1 complex acts as a novel modifier of myofilament function and localizes to the Z-disk in cardiac myocytes.

Contractile perturbations downstream of Ca(2+) binding to troponin C, the so-called sarcomere-controlled mechanisms, represent the earliest indicators of energy remodeling in the diseased heart [1]. Central to cellular energy "sensing" is the adenosine monophosphate-activated kinase (AMPK) pathway, which is known to directly target myofilament proteins and alter contractility [2-6]. We previously showed that the upstream AMPK kinase, LKB1/MO25/STRAD, impacts myofilament function independently of AMPK [5]. Therefore, we hypothesized that the LKB1 complex associated with myofilament proteins and that alterations in energy signaling modulated targeting or localization of the LKB1 complex to the myofilament. Using an integrated strategy of myofilament mechanics, immunoblot analysis, co-immunoprecipitation, mass spectroscopy, and immunofluorescence, we showed that 1) LKB1 and MO25 associated with myofibrillar proteins, 2) cellular energy stress re-distributed AMPK/LKB1 complex proteins within the sarcomere, and 3) the LKB1 complex localized to the Z-Disk and interacted with cytoskeletal and energy-regulating proteins, including vinculin and ATP Synthase (Complex V). These data represent a novel role for LKB1 complex proteins in myofilament function and myocellular "energy" sensing in the heart.

LKB1/Mo25/STRAD uniquely impacts sarcomeric contractile function and posttranslational modification.

The myocardium undergoes extensive metabolic and energetic remodeling during the progression of cardiac disease. Central to remodeling are changes in the adenine nucleotide pool. Fluctuations in these pools can activate AMP-activated protein kinase (AMPK), the central regulator of cellular energetics. Binding of AMP to AMPK not only allosterically activates AMPK but also promotes phosphorylation of AMPK by an upstream kinase complex, LKB1/Mo25/STRAD (liver kinase B 1, mouse protein 25, STE-related adaptor protein). AMPK phosphorylation by the LKB1 complex results in a substantial increase in AMPK activity. Molecular targeting by the LKB1 complex depends on subcellular localization and transcriptional expression. Yet, little is known about the ability of the LKB1 complex to modulate targeting of AMPK after activation. Accordingly, we hypothesized that differing stoichiometric ratios of LKB1 activator complex to AMPK would uniquely impact myofilament function. Demembranated rat cardiac trabeculae were incubated with varying ratios of the LKB1 complex to AMPK or the LKB1 complex alone. After incubation, we measured the Ca(2+) sensitivity of tension, rate constant for tension redevelopment, maximum tension generation, length-dependent activation, cooperativity, and sarcomeric protein phosphorylation status. We found that the Ca(2+) sensitivity of tension and cross-bridge dynamics were dependent on the LKB1 complex/AMPK ratio. We also found that the LKB1 complex desensitizes and suppresses myofilament function independently of AMPK. A phospho-proteomic analysis of myofilament proteins revealed site-specific changes in cardiac Troponin I (cTnI) phosphorylation, as well as a unique distribution of cTnI phosphospecies that were dependent on the LKB1 complex/ AMPK ratio. Fibers treated with the LKB1 complex alone did not alter cTnI phosphorylation or phosphospecies distribution. However, LKB1 complex treatment independent of AMPK increased phosphorylation of myosin-binding protein C. Therefore, we conclude that the LKB1/AMPK signaling axis is able to alter muscle function through multiple mechanisms.

Diet and sex modify exercise and cardiac adaptation in the mouse.

The heart adapts to exercise stimuli in a sex-dimorphic manner when mice are fed the traditional soy-based chow. Females undergo more voluntary exercise (4 wk) than males and exhibit more cardiac hypertrophy per kilometer run (18, 32). We have found that diet plays a critical role in cage wheel exercise and cardiac adaptation to the exercise stimulus in this sex dimorphism. Specifically, feeding male mice a casein-based, soy-free diet increases daily running distance over soy-fed counterparts to equal that of females. Moreover, casein-fed males have a greater capacity to increase their cardiac mass in response to exercise compared with soy-fed males. To further explore the biochemical mechanisms for these differences, we performed a candidate-based RT-PCR screen on genes previously implicated in diet- or exercise-based cardiac hypertrophy. Of the genes screened, many exhibit significant exercise, diet, or sex effects but only transforming growth factor-ß1 shows a significant three-way interaction with no genes showing a two-way interaction. Finally, we show that the expression and activity of adenosine monophosphate-activated kinase-α2 and acetyl-CoA carboxylase is dependent on exercise, diet, and sex.

ANG II-induced hypertension in the VCD mouse model of menopause is prevented by estrogen replacement during perimenopause.

Premenopausal females are resistant to the development of hypertension, and this protection is lost after the onset of menopause, resulting in a sharp increase in disease onset and severity. However, it is unknown how a fluctuating ovarian hormone environment during the transition from perimenopause to menopause impacts the onset of hypertension, and whether interventions during perimenopause prevent disease onset after menopause. A gradual transition to menopause was induced by repeated daily injections of 4-vinylcyclohexene diepoxide (VCD). ANG II (800 ng·kg(-1)·min(-1)) was infused into perimenopausal and menopausal female mice for 14 days. A separate cohort of mice received 17ß-estradiol replacement during perimenopause. ANG II infusion produced significantly higher mean arterial pressure (MAP) in menopausal vs. cycling females, and 17ß-estradiol replacement prevented this increase. In contrast, MAP was not significantly different when ANG II was infused into perimenopausal and cycling females, suggesting that female resistance to ANG II-induced hypertension is intact during perimenopause. ANG II infusion caused a significant glomerular hypertrophy, and hypertrophy was not impacted by hormonal status. Expression levels of aquaporin-2 (AQP2), a collecting duct protein, have been suggested to reflect blood pressure. AQP2 protein expression was significantly downregulated in the renal cortex of the ANG II-infused menopause group, where blood pressure was increased. AQP2 expression levels were restored to control levels with 17ß-estradiol replacement. This study indicates that the changing hormonal environment in the VCD model of menopause impacts the severity of ANG II-induced hypertension. These data highlight the utility of the ovary-intact VCD model of menopause as a clinically relevant model to investigate the physiological mechanisms of hypertension that occur in women during the transition into menopause.

Synergistic Effect of Transmyocardial Revascularization and Platelet-Rich Plasma on Improving Cardiac Function After Coronary Artery Bypass Grafting.

Background Coronary artery disease (CAD) is a global health burden, contributing to mortality and morbidity. A proportion of patients with CAD suffer from diffuse CAD, where conventional revascularization techniques such as percutaneous coronary intervention and coronary artery bypass grafting (CABG) may be insufficient to adequately restore myocardial perfusion. Transmyocardial revascularization (TMR) uses a laser to create microscopic channels in the myocardium, inducing inflammation, angiogenesis, and neovascularization to improve perfusion to ischemic regions. Platelet-rich plasma (PRP) is an autologous concentrate of platelets that contains a myriad of growth factors and bioactive proteins, which have been shown to promote tissue regeneration and wound healing. The combination of TMR and PRP therapies has been proposed to synergistically enhance myocardial revascularization and functional recovery in patients with advanced CAD undergoing surgical revascularization. Methods This study evaluated the efficacy of combining TMR and PRP with CABG in improving cardiac function in diffuse CAD patients. Fifty-two patients were randomized to CABG alone (n = 16), CABG+TMR (n = 17), CABG+PRP (n = 10), and CABG+TMR+PRP (n = 9). TMR was performed using a holmium:YAG laser to create 10 channels in the inferolateral left ventricular wall. PRP was prepared from autologous whole blood and injected into the myocardium adjacent to the TMR channels. Cardiac function was assessed using speckle-tracking echocardiography preoperatively, postoperatively, and at one-year follow-up. Adverse events, including post-operative atrial fibrillation, acute kidney injury, and readmissions, were also recorded. Statistical analyses were performed to compare outcomes between the treatment groups. Results The CABG+TMR+PRP group showed significantly improved global longitudinal strain (GLS) at one year compared to CABG alone (mean GLS -15.96 vs -12.09, p = 0.02). Post-operative left ventricular ejection fraction trended higher in the TMR+PRP group (57.78%) vs other groups, but not significantly. Post-operative atrial fibrillation was higher in the TMR+PRP group (67% vs 25%, p = 0.04), potentially reflecting increased inflammation. No significant differences were observed in other adverse events. Conclusions The results of this study suggest a synergistic benefit of combining TMR and PRP therapies as an adjunct to CABG in patients with diffuse CAD. The significant improvement in GLS at one year in the TMR+PRP group compared to CABG alone indicates enhanced myocardial remodeling and functional recovery, which may translate to improved long-term outcomes. The higher incidence of postoperative atrial fibrillation in the TMR+PRP group warrants further investigation but may reflect the heightened inflammatory response necessary for angiogenesis and tissue regeneration. Prospective, randomized controlled trials with larger sample sizes and longer follow-up periods are needed to validate these findings and optimize treatment protocols. Nonetheless, concomitant TMR+PRP therapy represents a promising approach to augmenting myocardial revascularization and recovery in patients with advanced CAD undergoing surgical revascularization.

Sexually dimorphic myofilament function and cardiac troponin I phosphospecies distribution in hypertrophic cardiomyopathy mice.

The pathological progression of hypertrophic cardiomyopathy (HCM) is sexually dimorphic such that male HCM mice develop phenotypic indicators of cardiac disease well before female HCM mice. Here, we hypothesized that alterations in myofilament function underlies, in part, this sex dimorphism in HCM disease development. Firstly, 10-12month female HCM (harboring a mutant [R403Q] myosin heavy chain) mice presented with proportionately larger hearts than male HCM mice. Next, we determined Ca(2+)-sensitive tension development in demembranated cardiac trabeculae excised from 10-12month female and male HCM mice. Whereas HCM did not impact Ca(2+)-sensitive tension development in male trabeculae, female HCM trabeculae were more sensitive to Ca(2+) than wild-type (WT) counterparts and both WT and HCM males. We hypothesized that the underlying cause of this sex difference in Ca(2+)-sensitive tension development was due to changes in Ca(2+) handling and sarcomeric proteins, including expression of SR Ca(2+) ATPase (2a) (SERCA2a), ß-myosin heavy chain (ß-MyHC) and post-translational modifications of myofilament proteins. Female HCM hearts showed an elevation of SERCA2a and ß-MyHC protein whereas male HCM hearts showed a similar elevation of ß-MyHC protein but a reduced level of cardiac troponin T (cTnT) phosphorylation. We also measured the distribution of cardiac troponin I (cTnI) phosphospecies using phosphate-affinity SDS-PAGE. The distribution of cTnI phosphospecies depended on sex and HCM. In conclusion, female and male HCM mice display sex dimorphic myofilament function that is accompanied by a sex- and HCM-dependent distribution of sarcomeric proteins and cTnI phosphospecies.

PNA5, A Novel Mas Receptor Agonist, Improves Neurovascular and Blood-Brain-Barrier Function in a Mouse Model of Vascular Cognitive Impairment and Dementia.

It is well established that decreased brain blood flow, increased reactive oxygen species production (ROS), and pro-inflammatory mechanisms accelerate neurodegenerative disease progressions, including vascular cognitive impairment and dementia (VCID). Previous studies in our laboratory have shown that our novel glycosylated Angiotensin-(1-7) Mas receptor agonist PNA5 reverses cognitive deficits, decreases ROS production, and inhibits inflammatory cytokine production in our preclinical mouse model of VCID that is induced by chronic heart failure (VCID-HF). In the present study, the effects of VCID-HF and treatment with PNA5 on microglia activation, blood-brain-barrier (BBB) integrity, and neurovascular coupling were assessed in our mouse model of VCID-HF. Three-month-old male C57BL/6J mice were subjected to myocardial infarction (MI) to induce heart failure for four weeks and then treated with subcutaneous injections of extended-release PNA5. Microglia activation, BBB permeability, cerebral perfusion, and neurovascular coupling were assessed. Results show that in our VCID-HF model, there was an increase in microglial activation and recruitment within the CA1 and CA3 regions of the hippocampus, a disruption in BBB integrity, and a decrease in neurovascular coupling. Treatment with PNA5 reversed these neuropathological effects of VCID-HF, suggesting that PNA5 may be an effective disease-modifying therapy to treat and prevent VCID. This study identifies potential mechanisms by which heart failure may induce VCID and highlights the possible mechanisms by which treatment with our novel glycosylated Angiotensin-(1-7) Mas receptor agonist, PNA5, may protect cognitive function in our model of VCID.

Epicardial placement of human placental membrane protects from heart injury in a swine model of myocardial infarction.

Cardiac ischemic reperfusion injury (IRI) is paradoxically instigated by reestablishing blood-flow to ischemic myocardium typically from a myocardial infarction (MI). Although revascularization following MI remains the standard of care, effective strategies remain limited to prevent or attenuate IRI. We hypothesized that epicardial placement of human placental amnion/chorion (HPAC) grafts will protect against IRI. Using a clinically relevant model of IRI, swine were subjected to 45 min percutaneous ischemia followed with (MI + HPAC, n = 3) or without (MI only, n = 3) HPAC. Cardiac function was assessed by echocardiography, and regional punch biopsies were collected 14 days post-operatively. A deep phenotyping approach was implemented by using histological interrogation and incorporating global proteomics and transcriptomics in nonischemic, ischemic, and border zone biopsies. Our results established HPAC limited the extent of cardiac injury by 50% (11.0 ± 2.0% vs. 22.0 ± 3.0%, p = 0.039) and preserved ejection fraction in HPAC-treated swine (46.8 ± 2.7% vs. 35.8 ± 4.5%, p = 0.014). We present comprehensive transcriptome and proteome profiles of infarct (IZ), border (BZ), and remote (RZ) zone punch biopsies from swine myocardium during the proliferative cardiac repair phase 14 days post-MI. Both HPAC-treated and untreated tissues showed regional dynamic responses, whereas only HPAC-treated IZ revealed active immune and extracellular matrix remodeling. Decreased endoplasmic reticulum (ER)-dependent protein secretion and increased antiapoptotic and anti-inflammatory responses were measured in HPAC-treated biopsies. We provide quantitative evidence HPAC reduced cardiac injury from MI in a preclinical swine model, establishing a potential new therapeutic strategy for IRI. Minimizing the impact of MI remains a central clinical challenge. We present a new strategy to attenuate post-MI cardiac injury using HPAC in a swine model of IRI. Placement of HPAC membrane on the heart following MI minimizes ischemic damage, preserves cardiac function, and promotes anti-inflammatory signaling pathways.

Glycosylated Ang-(1-7) MasR Agonist Peptide Poly Lactic-co-Glycolic Acid (PLGA) Nanoparticles and Microparticles in Cognitive Impairment: Design, Particle Preparation, Physicochemical Characterization, and In Vitro Release.

Heart failure (HF) causes decreased brain perfusion in older adults, and increased brain and systemic inflammation increases the risk of cognitive impairment and Alzheimer's disease (AD). Glycosylated Ang-(1-7) MasR agonists (PNA5) has shown improved bioavailability, stability, and brain penetration compared to Ang-(1-7) native peptide. Despite promising results and numerous potential applications, clinical applications of PNA5 glycopeptide are limited by its short half-life, and frequent injections are required to ensure adequate treatment for cognitive impairment. Therefore, sustained-release injectable formulations of PNA5 glycopeptide are needed to improve its bioavailability, protect the peptide from degradation, and provide sustained drug release over a prolonged time to reduce injection administration frequency. Two types of poly(D,L-lactic-co-glycolic acid) (PLGA) were used in the synthesis to produce nanoparticles (≈0.769−0.35 µm) and microparticles (≈3.7−2.4 µm) loaded with PNA5 (ester and acid-end capped). Comprehensive physicochemical characterization including scanning electron microscopy, thermal analysis, molecular fingerprinting spectroscopy, particle sizing, drug loading, encapsulation efficiency, and in vitro drug release were conducted. The data shows that despite the differences in the size of the particles, sustained release of PNA5 was successfully achieved using PLGA R503H polymer with high drug loading (% DL) and high encapsulation efficiency (% EE) of >8% and >40%, respectively. While using the ester-end PLGA, NPs showed poor sustained release as after 72 h, nearly 100% of the peptide was released. Also, lower % EE and % DL values were observed (10.8 and 3.4, respectively). This is the first systematic and comprehensive study to report on the successful design, particle synthesis, physicochemical characterization, and in vitro glycopeptide drug release of PNA5 in PLGA nanoparticles and microparticles.

Antagonism of the mu-delta opioid receptor heterodimer enhances opioid antinociception by activating Src and calcium/calmodulin-dependent protein kinase II signaling.

ABSTRACT: The opioid receptors are important regulators of pain, reward, and addiction. Limited evidence suggests the mu and delta opioid receptors form a heterodimer (MDOR), which may act as a negative feedback brake on opioid-induced analgesia. However, evidence for the MDOR in vivo is indirect and limited, and there are few selective tools available. We recently published the first MDOR-selective antagonist, D24M, allowing us to test the role of the MDOR in mice. We thus cotreated CD-1 mice with D24M and opioids in tail flick, paw incision, and chemotherapy-induced peripheral neuropathy pain models. D24M treatment enhanced oxymorphone antinociception in all models by 54.7% to 628%. This enhancement could not be replicated with the mu and delta selective antagonists CTAP, naltrindole, and naloxonazine, and D24M had a mild transient effect in the rotarod test, suggesting this increase is selective to the MDOR. However, D24M had no effect on morphine or buprenorphine, suggesting that only specific opioids interact with the MDOR. To find a mechanism, we performed phosphoproteomic analysis on brainstems of mice. We found that the kinases Src and CaMKII were repressed by oxymorphone, which was restored by D24M. We were able to confirm the role of Src and CaMKII in D24M-enhanced antinociception using small molecule inhibitors (KN93 and Src-I1). Together, these results provide direct in vivo evidence that the MDOR acts as an opioid negative feedback brake, which occurs through the repression of Src and CaMKII signal transduction. These results further suggest that MDOR antagonism could be a means to improve clinical opioid therapy.

Remodeling the cardiac transcriptional landscape with diet.

The perception that soy food products and dietary supplements will have beneficial effects on cardiovascular health has led to a massive consumer market. However, we have previously noted that diet profoundly affects disease progression in a genetic model of hypertrophic cardiomyopathy (HCM). In this model, a soy-based diet negatively impacts cardiac function in male mice. Given the frequent connection between functional changes and transcriptional changes, we investigated the effect of diet (soy- vs. milk-based) on cardiac gene expression and how it is affected by the additional factors of sex and disease. We found that gene expression in the heart is altered more by diet than by sex or an inherited disease. We also found that the healthy male heart may be sensitized to dietary perturbations of gene expression in that it displays a gene expression profile more similar to diseased male and female hearts than to healthy female hearts. These observations may in part account for documented divergence in HCM phenotypes between males and females and between diets.

Inhibition of ovarian KIT phosphorylation by the ovotoxicant 4-vinylcyclohexene diepoxide in rats.

In vitro exposure of Postnatal Day 4 (PND4) rat ovaries to the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys specifically primordial and primary follicles via acceleration of atresia. Because oocyte-expressed c-kit (KIT) plays a critical role in follicle survival and activation, a direct interaction of VCD with KIT as its mechanism of ovotoxicity was investigated. PND4 rat ovaries were cultured with and without VCD (30 µM) for 2 days. When assessed by Western analysis or mobility shift detection, phosphorylated KIT (pKIT) was decreased (P < 0.05) by VCD exposure, while total KIT protein was unaffected. Anti-mouse KIT2 (ACK2) antibody binds KIT and blocks its signaling pathways, whereas anti-mouse KIT 4 (ACK4) antibody binds KIT but does not block its activity. PND4 rat ovaries were incubated for 2 days with and without VCD with and without ACK2 (80 µg/ml) or ACK4 (80 µg/ml). ACK2 decreased pKIT; however, ACK4 had no effect. Conversely, ACK2 did not affect a VCD-induced decrease in pKIT, whereas ACK4 further reduced it. Because ACK2 and ACK4 (known to directly bind KIT) affect VCD responses, these results support the fact that VCD interacts directly with KIT. The effect of these antibodies on VCD-induced follicle loss was measured after 8 days of incubation. ACK2 further reduced (P < 0.05) VCD-induced follicle loss, whereas ACK4 did not affect it. These findings demonstrate that VCD induces ovotoxicity by direct inhibition of KIT autophosphorylation of the oocyte. The data also further support the vital function of KIT and its signaling pathway in primordial follicle survival and activation, as well as its role in VCD-induced ovotoxicity.