RNA sequencing of formalin fixed paraffin-embedded heart tissue provides transcriptomic information about chemotherapy-induced cardiotoxicity.

Gene expression of formalin-fixed paraffin-embedded (FFPE) tissue may serve for molecular studies on cardiovascular diseases. Chemotherapeutics, such as doxorubicin (DOX) may cause heart injury, but the mechanisms of these side effects of DOX are not well understood. This study aimed to investigate whether DOX-induced gene expression in archival FFPE heart tissue in experimental rats would correlate with the gene expression in fresh-frozen heart tissue by applying RNA sequencing technology. The results showed RNA from FFPE samples was degraded, resulting in a lower number of uniquely mapped reads. However, DOX-induced differentially expressed genes in FFPE were related to molecular mechanisms of DOX-induced cardiotoxicity, such as inflammation, calcium binding, endothelial dysfunction, senescence, and cardiac hypertrophy signaling. Our data suggest that, despite the limitations, RNA sequencing of archival FFPE heart tissue supports utilizing FFPE tissues from retrospective studies on cardiovascular disorders, including DOX-induced cardiotoxicity.

Deletion of Interleukin-1ß Converting Enzyme Alters Mouse Cardiac Structure and Function.

Interleukin-1ß converting enzyme (ICE, caspase-1) is a thiol protease that cleaves the pro-inflammatory cytokine precursors of IL-1ß and IL-18 into active forms. Given the association between caspase-1 and cardiovascular pathology, we analyzed the hearts of ICE knockout (ICE KO) mice to test the hypothesis that caspase-1 plays a significant role in cardiac morphology and function. We characterized the histological and functional changes in the hearts of ICE KO mice compared to the Wild type. The cardiomyocytes from the neonatal ICE KO mice showed an impaired response to oxidative stress. Subsequently, the hearts from the ICE KO mice were hypertrophied, with a significant increase in the left ventricular and septal wall thickness and a greater LV mass/body weight ratio. The ICE KO mice hearts exhibited irregular myofibril arrangements and disruption of the cristae in the mitochondrial structure. Proapoptotic proteins that were significantly increased in the hearts of ICE KO versus the Wild type included pErk, pJNK, p53, Fas, Bax, and caspase 3. Further, the antiapoptotic proteins Bag-1 and Bcl-2 are activated in ICE KO hearts. Functionally, there was an increase in the left ventricular epicardial diameter and volume in ICE KO. In conclusion, our findings support the important role of caspase-1 in maintaining cardiac health; specifically, a significant decrease in caspase-1 is detrimental to the cardiovascular system.

Short-Term Ingestion of Essential Amino Acid Based Nutritional Supplements or Whey Protein Improves the Physical Function of Older Adults Independently of Gut Microbiome.

SCOPE: Dietary proteins and essential amino acids (EAAs) are the major nutritional supplements that support the growth and activity of gut microbes contributing to the wellbeing of their host. This study hypothesizes that daily supplementation of the diet with either EAAs or whey protein for 12 weeks would improve the gut microbiome of older adults. METHODS AND RESULTS: The stool samples are processed and subjected to Illumina-based 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing. In both groups, the most abundant families are found in order of relative abundance included: Bacteroidaceae, Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Rikenellaceae, Enterobacteriaceae, Oscillospiraceae, Tannerellaceae, and Akkermansiaceae, which indicate that these subjects are able to maintain a same healthy microbial diversity in their guts. A significant finding is a reduction of proinflammatory cytokine, interleukin-18 (IL-18) in the EAAs group. It also uses the standard 6-min walking test (6MWT) as a measure of cardiopulmonary fitness. At the end of the study, the subjects in the EAAs group perform significantly better in the 6MWT as compared to the whey group. CONCLUSION: It seems plausible that the improved physical performance and reduced proinflammatory cytokine, IL-18 seen in the EAAs group, are independent of changes in gut microbiota.

Proteomic analysis of P. gingivalis-Lipopolysaccharide induced neuroinflammation in SH-SY5Y and HMC3 cells.

Chronic periodontitis and its keystone pathogen, Porphyromonas gingivalis, have increasingly been linked with Alzheimer's disease (AD). However, P.gingivalis-lipopolysaccharide (LPS) mediated release of neuroinflammatory proteins contributes to AD remains underexplored. In this study, we utilized data-independent acquisition mass spectrometry to characterize P.gingivalis-LPS induced profile of differentially expressed proteins associated with the neuroinflammatory response in human neuroblastoma (SH-SY5Y) and human microglial (HMC3) cells. We reported a set of 124 proteins in SH-SY5Y cells and 96 proteins in HMC3 cells whose levels were significantly upregulated or downregulated by exposure to P. gingivalis-LPS. Our findings demonstrate that P. gingivalis-LPS contributed to the elevated expressions of dementia biomarkers and pro-inflammatory cytokines that include APP, Aß1-42, Aß1-40, T-Tau, p-Tau, VEGF, TGF-ß, IL-1ß, IL-6 and TNF-α through 2 distinct pathways of extracellular sensing by cell surface receptors and intracellular cytosolic receptors. Interestingly, intracellular signaling proteins activated with P. gingivalis-LPS transfection using Lipofectamine™ 2000 had significantly higher fold change protein expression compared to the extracellular signaling with P. gingivalis-LPS treatment. Additionally, we also explored P. gingivalis-LPS mediated activation of caspase-4 dependent non canonical inflammasome pathway in both SH-SY5Y and HMC3 cells. In summary, P. gingivalis-LPS induced neuroinflammatory protein expression in SH-SY5Y and HMC3 cells, provided insights into the specific inflammatory pathways underlying the potential link between P. gingivalis-LPS infection and the pathogenesis of Alzheimer's disease and related dementias.

Inhibitors of Rho/MRTF/SRF Transcription Pathway Regulate Mitochondrial Function.

RhoA-regulated gene transcription by serum response factor (SRF) and its transcriptional cofactor myocardin-related transcription factors (MRTFs) signaling pathway has emerged as a promising therapeutic target for pharmacological intervention in multiple diseases. Altered mitochondrial metabolism is one of the major hallmarks of cancer, therefore, this upregulation is a vulnerability that can be targeted with Rho/MRTF/SRF inhibitors. Recent advances identified a novel series of oxadiazole-thioether compounds that disrupt the SRF transcription, however, the direct molecular target of these compounds is unclear. Herein, we demonstrate the Rho/MRTF/SRF inhibition mechanism of CCG-203971 and CCG-232601 in normal cell lines of human lung fibroblasts and mouse myoblasts. Further studies investigated the role of these molecules in targeting mitochondrial function. We have shown that these molecules hyperacetylate histone H4K12 and H4K16 and regulate the genes involved in mitochondrial function and dynamics. These small molecule inhibitors regulate mitochondrial function as a compensatory mechanism by repressing oxidative phosphorylation and increasing glycolysis. Our data suggest that these CCG molecules are effective in inhibiting all the complexes of mitochondrial electron transport chains and further inducing oxidative stress. Therefore, our present findings highlight the therapeutic potential of CCG-203971 and CCG-232601, which may prove to be a promising approach to target aberrant bioenergetics.

Valine improves mitochondrial function and protects against oxidative stress.

Among the branched-chain amino acids, leucine and isoleucine have been well studied for their roles in improving mitochondrial function and reducing oxidative stress. However, role of valine in mitochondrial function regulation and oxidative stress management remains elusive. This study investigated valine effect on mitochondrial function and oxidative stress in vitro. Valine increased expression of genes involved in mitochondrial biogenesis and dynamics. It upregulates mitochondrial function at complexes I, II, and IV levels of electron transport chain. Flow cytometry studies revealed, valine reduced oxidative stress by significantly lowering mitochondrial reactive oxygen species and protein expression of 4-hydroxynonenal. Functional role of valine against oxidative stress was analyzed by XFe96 Analyzer. Valine sustained oxidative phosphorylation and improved ATP generation rates during oxidative stress. In conclusion, our findings shed more light on the critical function of valine in protecting mitochondrial function thereby preventing mitochondrial/cellular damage induced by oxidative stress.

The Importance of Dopamine Deficiency Evaluation in Non-Alzheimer Disease Dementias.

BACKGROUND Dementia with Lewy bodies (DLB) is a common cause of dementia. Given the similarities between the symptoms of DLB and non-DLB Alzheimer disease (AD) and related dementias, patients can sometimes be misdiagnosed with AD. To increase the sensitivity of current DLB guidelines, the DLB Consortium published its fourth revised report in 2017 with increased diagnostic weight given to dopamine transporter (DAT) uptake in the basal ganglia, demonstrated by single-photon emission computed tomography or positron emission tomography imaging. We aimed to describe the role of DAT scans in evaluating dopamine deficiency in patients with overlapping symptoms of AD and DLB. CASE REPORT We present case studies of 3 patients with memory impairment who had a diagnosis of probable AD and were being treated with cholinesterase inhibitors. During treatment, dopamine deficiency was suspected and DAT scans were performed. All 3 patients revealed severe DAT deficits in the bilateral corpus striatum. These results were consistent with probable DLB as per the current revised DLB Consortium report. All patients received treatment with carbidopa/levodopa and demonstrated improved overall function. CONCLUSIONS All 3 of our cases demonstrated the role of DAT scans in evaluating dopamine deficiency syndromes in patients with overlapping symptoms of neurocognitive disorders. Thus, a DAT scan is critical for establishing an earlier and more definitive diagnosis of DLB, which provides treatment options for dopamine replacement. It also assists providers with prognostication of dopamine deficiency syndromes and is therefore beneficial in counseling patients and caregivers.

P. gingivalis-LPS Induces Mitochondrial Dysfunction Mediated by Neuroinflammation through Oxidative Stress.

Porphyromonas gingivalis (P. gingivalis), a key pathogen in periodontitis, is associated with neuroinflammation. Periodontal disease increases with age; 70.1% of adults 65 years and older have periodontal problems. However, the P. gingivalis- lipopolysaccharide (LPS)induced mitochondrial dysfunction in neurodegenerative diseases remains elusive. In this study, we investigated the possible role of P. gingivalis-LPS in mitochondrial dysfunction during neurodegeneration. We found that P. gingivalis-LPS treatment activated toll-like receptor (TLR) 4 signaling and upregulated the expression of Alzheimer's disease-related dementia and neuroinflammatory markers. Furthermore, the LPS treatment significantly exacerbated the production of reactive oxygen species and reduced the mitochondrial membrane potential. Our study highlighted the pivotal role of P. gingivalis-LPS in the repression of serum response factor (SRF) and its co-factor p49/STRAP that regulate the actin cytoskeleton. The LPS treatment repressed the genes involved in mitochondrial function and biogenesis. P. gingivalis-LPS negatively altered oxidative phosphorylation and glycolysis and reduced total adenosine triphosphate (ATP) production. Additionally, it specifically altered the mitochondrial functions in complexes I, II, and IV of the mitochondrial electron transport chain. Thus, it is conceivable that P. gingivalis-LPS causes mitochondrial dysfunction through oxidative stress and inflammatory events in neurodegenerative diseases.

Differential plasma protein expression after ingestion of essential amino acid-based dietary supplement verses whey protein in low physical functioning older adults.

In a recent randomized, double-blind, placebo-controlled trial, we were able to demonstrate the superiority of a dietary supplement composed of essential amino acids (EAAs) over whey protein, in older adults with low physical function. In this paper, we describe the comparative plasma protein expression in the same subject groups of EAAs vs whey. The plasma proteomics data was generated using SOMA scan assay. A total of twenty proteins were found to be differentially expressed in both groups with a 1.5-fold change. Notably, five proteins showed a significantly higher fold change expression in the EAA group which included adenylate kinase isoenzyme 1, casein kinase II 2-alpha, Nascent polypeptide-associated complex subunit alpha, peroxiredoxin-1, and peroxiredoxin-6. These five proteins might have played a significant role in providing energy for the improved cardiac and muscle strength of older adults with LPF. On the other hand, fifteen proteins showed slightly lower fold change expression in the EAA group. Some of these 15 proteins regulate metabolism and were found to be associated with inflammation or other comorbidities. Gene Ontology (GO) enrichment analysis showed the association of these proteins with several biological processes. Furthermore, protein-protein interaction network analysis also showed distinct networks between upregulated and downregulated proteins. In conclusion, the important biological roles of the upregulated proteins plus better physical function of participants in the EAAs vs whey group demonstrated that EAAs have the potential to improve muscle strength and physical function in older adults. This study was registered with ClinicalTrials.gov: NCT03424265 "Nutritional interventions in heart failure."

Rho/SRF Inhibitor Modulates Mitochondrial Functions.

CCG-1423 is a Rho A pathway inhibitor that has been reported to inhibit Rho/SRF-mediated transcriptional regulation. Serum response factor and its cofactors, which include ternary complex factors and myocardin-related transcription factors, regulate various cellular functions. In this study, we observed that CCG-1423 modulates the mitochondrial functions. The effect of this small molecule drug was determined by measuring mitochondrial function using an XFe96 Analyzer and an Oxygraph 2k (O2k) high-resolution respirometer. CCG-1423 treatment significantly reduced oxidative phosphorylation in a dose-dependent manner. However, CCG-1423 increased the glycolytic rate. We also observed that histone 4 at lysine-16 underwent hyperacetylation with the treatment of this drug. Immunolabeling with F-actin and MitoTracker revealed the alteration in the actin cytoskeleton and mitochondria. Taken together, our findings highlight a critical role of CCG-1423 in inhibiting the transcription of SRF/p49 and PGC-1α, ß, resulting in the downregulation of mitochondrial genes, leading to the repression of mitochondrial oxidative phosphorylation and overall ATP reduction. This study provides a better understanding of the effects of CCG-1423 on mitochondria, which may be useful for the assessment of the potential clinical application of CCG-1423 and its derivatives.

Disparity and Multimorbidity in Heart Failure Patients Over the Age of 80.

Background: Healthcare is currently struggling to provide access and coverage for an increasingly diverse aging population who frequently have multiple co-morbid conditions complicating their care and medical management. Methods: This retrospective study analyzed the prevalence and distribution of common co-morbid conditions (hypertension, dyslipidemia, dementia, and diabetes mellitus) in 316 elderly heart failure patients (age range 80-103; mean 87 ±4.9). Results: Chart review analysis showed a racial distribution of 65 African American versus 251 Caucasian patients (21 vs. 79%). Hypertension was comparable in both groups (98.5% African American vs. 92.4% Caucasian). Dyslipidemia, diabetes and dementia diagnoses were all approximately 20% higher in African American versus Caucasian patients. The concurrent presence of all four conditions was approximately three times more prevalent in African Americans (18.5%) versus Caucasians (7.2%). Conclusion: Our study is unique for studying disparity in octogenarian and nonagenarians residing in a rural setting. Our results also highlight the importance of making a special effort to engage older African American patients in seeking healthcare. In addition, strategies must be designed to reduce barriers that impede access and availability of resources and clinical care, especially in economically underserved regions of the country.

Quality Improvement in Delirium Health Literacy in Older Adult Patients and Their Caregivers Attending a Geriatric Clinic.

Background: Delirium is a common medical condition that is highly prevalent in older adults who are at increased risk for its development with any illness, post-surgery or during hospitalization. The purpose of our study was to evaluate the health literacy of older adult patients and their caregivers about delirium, offer a brief educational intervention, and reevaluate their knowledge post intervention. Materials and Methods: We conducted a quality improvement project, focused on delirium health literacy in older adult patients ≥60 years and their caregivers. Delirium knowledge of participants was evaluated in a pre-education survey after which they were given a delirium education booklet to read. A post-education delirium survey was conducted within 2-3 weeks of the educational intervention. Chi-square test was used to analyze the knowledge base of older adults. Results: The study population consisted of a total of 70 older adults who participated in pre-education (n=35) and post-education (n=35) surveys. Older adult patients and their caregivers had significant knowledge gaps about the potential causes or etiologies, risk factors, symptomatology, and prevention of delirium in the pre-education survey. After the educational intervention, in the post-education survey, there were overall improvements in knowledge base of older adults in differentiating delirium with dementia (43% vs 94%, p<0.01) recognizing signs and symptoms (77% vs 94%, p<0.05), complications (76% vs 100%, p<0.01) and identifying the etiological factors associated with delirium. Conclusion: The quality improvement project demonstrated that older adults and caregivers have significant knowledge deficits about the common condition of delirium. This study also demonstrated that older adults were able to improve their health literacy regarding delirium after the intervention. Appropriate education on delirium for patients and caregivers might help in earlier identification, prevention, and better overall management of delirium.

Does Superior Bone Health Promote a Longer Lifespan?

INTRODUCTION: Public health achievements throughout the last century have resulted in a steady increase in life expectancy. An emergent subset has distinguished themselves, living well beyond the ninth decade by avoiding or delaying the onset of most age-related diseases, including bone diseases and fractures. In this study, we evaluated the bone health of the oldest community-dwelling individuals living in rural Arkansas. METHODS: 299 patients aged ≥90 years were retrospectively reviewed for recorded fractures within 12 years prior to the investigation period. Records were also examined for medications and test results pertinent to bone health, including thyroid stimulating hormone, vitamin D levels, hematocrit, hemoglobin, body mass index, and bone densitometric values. RESULTS: 68 patients (23%) had at least one fracture documented, and 15 had >1 fracture. 40% of patients with fractures had osteoporosis and 28% had osteopenia, respectively. 232 patients (78%) had no documented fractures, and of these, only 18% had osteoporosis and 16% had osteopenia. No significant clinical markers were found among the very old to explain the relatively low occurrence of fractures. CONCLUSIONS: Patients over 90 years of age had an overall low prevalence of fractures and relative preservation of bone health, suggesting a preserved bone molecular profile in these individuals. Epigenetic factors and activity levels might also have favorably affected bone health. The low percentage of osteoporosis and fractures likely reduced the morbidity and mortality in this population, potentially contributing to their overall longevity.

Cardiovascular Health in Individuals with Exceptional Longevity Residing in Arkansas.

Cardiovascular disease is a common comorbidity associated with an aging population. However, there is a unique group of individuals whose age-defying qualities are still being investigated. This retrospective chart review analyzed various cardiac and metabolic health parameters to characterize the prevalence of heart failure and metabolic derangements in individuals aged 90 years old or older in central Arkansas. Only 236 of the 291 patients in our study cohort had blood pressures recorded. Of these, 50% had systolic blood pressures ≥140 mmHg. Additionally, 77% had pulse pressures ≥50 mmHg. Of the 96 patients with BNP data, 44% had values ≥300 pg/mL. There was a slight positive correlation between aging and HDL cholesterol, while there was a negative correlation between aging and both total cholesterol and LDL cholesterol. A majority of our patients had both elevated systolic blood pressures and elevated pulse pressures. A majority also had high BNP values, indicative of some degree of heart failure. Additionally, atrial fibrillation was a common arrhythmia identified on EKG. However, these oldest of the old patients had fewer documented metabolic derangements. These findings lay important groundwork for further investigation into lifestyle and genetic components that allow them to live exceptionally long with such comorbidities.

Caregiver Burden: Caregiving Workshops Have a Positive Impact on Those Caring for Individuals With Dementia in Arkansas.

Millions of Americans live with dementia. Caregivers of this population provide countless hours of multifaceted, complex care that frequently cause unrelenting stress which can result in immense burden. However, it is not fully understood what efforts can be made to reduce the stress among caregivers of persons with dementia (PWD). Therefore, the aim of this pretest-posttest designed study was to evaluate changes in caregiver burden after providing an educational intervention to those caring for PWD in Arkansas. Forty-one participants completed the Zarit Caregiver Burden Scale before and after attending a 4-hour dementia-focused caregiving workshop. The analysis of the means, standard deviations, and paired t tests showed that there was an increase in the confidence and competence in caring for PWD 30 to 45 days after attending the workshop. Health care providers need to understand both the vital role caregivers provide in managing a PWD and the importance of the caregiver receiving education about their role as a caregiver. Utilizing caregiver educational programs is a first step.

Metabolic Evaluation of the Dietary Guidelines' Ounce Equivalents of Protein Food Sources in Young Adults: A Randomized Controlled Trial.

BACKGROUND: The Dietary Guidelines for Americans (DGAs) published an "ounce equivalents" recommendation to help consumers meet protein requirements with a variety of protein food sources. However, the metabolic equivalency of these varied protein food sources has not been established. OBJECTIVE: We have investigated the hypothesis that the anabolic responses to consumption of ounce equivalents of protein food sources would be directly related to the essential amino acid (EAA) content of the protein food source. METHODS: Following 3 d of dietary control, a total of 56 healthy young adults underwent an 8.5-h metabolic study using stable isotope tracer methodology. The changes from baseline following consumption of 1 of 7 different protein food sources were compared with the baseline value for that individual (n = 8 per group). RESULTS: Consumption of ounce equivalents of animal-based protein food sources (beef sirloin, pork loin, eggs) resulted in a greater gain in whole-body net protein balance above baseline than the ounce equivalents of plant-based protein food sources (tofu, kidney beans, peanut butter, mixed nuts; P < 0.01). The improvement in whole-body net protein balance was due to an increase in protein synthesis (P < 0.05) with all the animal protein sources, whereas the egg and pork groups also suppressed protein breakdown compared with the plant protein sources (P < 0.01). The magnitude of the whole-body net balance (anabolic) response was correlated with the EAA content of the protein food source (P < 0.001). CONCLUSION: The "ounce equivalents" of protein food sources as expressed in the DGAs are not metabolically equivalent in young healthy individuals. The magnitude of anabolic response to dietary proteins should be considered as the DGAs develop approaches to establish healthy eating patterns.

Alternative Splicing Increases Sirtuin Gene Family Diversity and Modulates Their Subcellular Localization and Function.

Alternative splicing generates multiple distinct isoforms that increase transcriptome and proteome diversity. There are seven sirtuin genes in humans, each consists of multiple exons that are likely to undergo alternative splicing. Our aim was to characterize the effect of alternative splicing on the sirtuin genes. Here, we report the identification of 23 human sirtuin isoforms, most of which were not previously reported. Five of the sirtuin genes had more than one isoform, whereas sirtuin-6 had nine isoforms. Exon skipping was the main event. Most of the sirtuin isoforms were deficient in parts of the protein domains, including the catalytic domain, the N- or C-terminus, nuclear localization signal or mitochondrial targeting signal. The domain loss caused potential structural changes. Three SIRT1 isoforms had a differential effect on the mitochondrial oxygen consumption rate. Age-related changes in the expression of SIRT1 isoforms were observed in the human heart in fetus, adults, and very old individuals. We also identified 15 sirtuin isoforms in mice. Our data indicate that alternative splicing increases sirtuin gene diversity and may modulate subcellular localization and function, thereby adding complexity to the gene regulation of mitochondrial respiration, metabolism, and cardiac function during maturation and aging.

Daily Consumption of a Specially Formulated Essential Amino Acid-Based Dietary Supplement Improves Physical Performance in Older Adults With Low Physical Functioning.

We have investigated the hypothesis that nutritional supplementation of the diet in low-physical-functioning older individuals with a specially formulated composition based on essential amino acids (EAAs) would improve physical function as compared to supplementation with the same amount of whey protein. A third group of comparable volunteers were given nutrition education but no supplementation of the diet. After 6 weeks of whey protein supplementation (n = 32), there was no effect on the distance walked in 6 minutes, but the distance walked improved significantly from the pre-value after 12 weeks of whey supplementation. EAA consumption (n = 28) significantly improved walking distance at both 6 and 12 weeks. The distance walked at 12 weeks (419.0 ± 25.0 m) was 35.4 m greater than the pre-value of 384.0 ± 23.0 m (p < .001). The increase in distance walked by the EAA group was also significantly greater than that in the whey group at both 6 and 12 weeks (p < .01). In contrast, a decrease in distance walked was observed in the control group (n = 32) (not statistically significant, NS). EAA supplementation also improved grip strength and leg strength, and decreased body weight and fat mass. Plasma low-density lipoprotein concentration was significantly reduced in the EAA group, as well as the concentration of macrophage migration inhibitory factor. There were no adverse responses in any groups, and compliance was greater than 95% in all individuals consuming supplements. We conclude that dietary supplementation with an EAA-based composition may be a beneficial therapy in older individuals with low physical functional capacity. Clinical Trials Registration Number: This study was registered with ClinicalTrials.gov: NCT03424265-"Nutritional interventions in heart failure."