Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging

Short-term rapamycin persistently improves cardiac function after cessation of treatment in aged male and female mice.

Thesis (Ph.D.)--University of Washington, 2017Cardiac aging is an intrinsic process that results in impaired cardiac function and dysregulation of cellular and molecular quality control mechanisms. These effects are evident in the decline of diastolic function, increase in left ventricular hypertrophy, metabolic substrate shifts, and alterations to the cardiac proteome. This thesis covers the quality control mechanisms that are associated with cardiac aging, results from an anti-aging intervention in aged mice, and a review of mitochondrial dysfunction in the heart. Chapter one is a review of the quality control mechanisms in aging myocardium. Chapter two consists of the results of several mouse experiments that compare the cardiac function, proteomes, and metabolomes of aged and young controls, along with rapamycin treated aged mice. The novelty of this study comes from the inclusion of a group of animals treated only transiently with the drug, then followed for eight weeks post-drug-removal. This persistence cohort may hold clues to deriving long-lasting benefits of rapamycin with only transient treatment. Chapter three includes more results from the cohorts used in chapter two, from work done by our collaborators in two laboratories at the University of Washington. Finally, chapter four is a review of the mechanisms and phenotypes of mitochondrial dysfunction in the aging heart. The goal of my thesis work is to test the persistence of the improvement of cardiac function by rapamycin treatment, and use the correlating changes in the cardiac proteome and metabolome to discover a novel mechanism of functional improvement of the heart in aged animals

Innate Immune Detection of Flagellin Positively and Negatively Regulates <i>Salmonella</i> Infection

<div><p><i>Salmonella enterica</i> serovar Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is required for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems for host defense against wild type <i>Salmonella</i> infection is complex, and innate immune detection of flagellin contributes to both protection and susceptibility. We hypothesized that efficient modulation of flagellin expression <i>in vivo</i> permits <i>Salmonella</i> to evade innate immune detection and limit the functional role of flagellin-specific host innate defenses. To test this hypothesis, we used <i>Salmonella</i> deficient in the anti-sigma factor <i>flgM</i>, which overproduce flagella and are attenuated <i>in vivo</i>. In this study we demonstrate that flagellin recognition by the innate immune system is responsible for the attenuation of <i>flgM⁻ S.</i> Typhimurium, and dissect the contribution of each flagellin recognition pathway to bacterial clearance and inflammation. We demonstrate that caspase-1 controls mucosal and systemic infection of <i>flgM⁻ S.</i> Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum and systemic infection, but attenuates intestinal inflammation. Our results indicate that <i>Salmonella</i> evasion of caspase-1 dependent flagellin recognition is critical for establishing infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition helps <i>Salmonella</i> induce intestinal inflammation and establish a niche in the inflamed gut.</p></div

Use of implicit persuasion in decision making about adjuvant cancer treatment: A potential barrier to shared decision making

Shared decision making (SDM) is widely advocated, especially for preference-sensitive decisions like those on adjuvant treatment for early-stage cancer. Here, decision making involves a subjective trade-off between benefits and side-effects, and therefore, patients' informed preferences should be taken into account. If clinicians consciously or unconsciously steer patients towards the option they think is in their patients' best interest (i.e. implicit persuasion), they may be unwittingly subverting their own efforts to implement SDM. We assessed the frequency of use of implicit persuasion during consultations and whether the use of implicit persuasion was associated with expected treatment benefit and/or decision making. Observational study design in which consecutive consultations about adjuvant systemic therapy with stage I-II breast cancer patients treated at oncology outpatient clinics of general teaching hospitals and university medical centres were audiotaped, transcribed and coded by two researchers independently. In total, 105 patients (median age = 59; range: 35-87 years) were included. A median of five (range: 2-10) implicitly persuasive behaviours were employed per consultation. The number of behaviours used did not differ by disease stage (P = 0.07), but did differ by treatment option presented (P = 0.002) and nodal status (P = 0.01). About 50% of patients with stage I or node-negative disease were steered towards undergoing chemotherapy, whereas 96% of patients were steered towards undergoing endocrine therapy, irrespective of expected treatment benefit. Decisions were less often postponed if more implicit persuasion was used (P = 0.03). Oncologists frequently use implicit persuasion, steering patients towards the treatment option that they think is in their patients' best interest. Expected treatment benefit does not always seem to be the driving force behind implicit persuasion. Awareness of one's use of these steering behaviours during decision making is a first step to help overcome the performance gap between advocating and implementing SD

Flagellin detection accounts for the caspase-1 increased susceptibility during acute mucosal infection in streptomycin-pretreated mice.

<p>Bacterial burden of WT C57BL/6 (n = 10) and caspase-1−/− (n = 16) mice infected with 1000 cfu flagellin<i>⁻ Salmonella</i> in the cecum (A), spleen (B), liver (C), MLN (D). Bacterial burden WT C57BL/6 (n = 15) and TLR5−/− (n = 16) mice infected with 1000 cfu flagellin<i>⁻ Salmonella</i> in the cecum (E), spleen (F), liver (G), MLN (H). Figures A–D are the combined data from two independent experiments. Figures E–H are the combined data from three independent experiments. Mann-Whitney test * = p<0.05. *** = p<0.001.</p

<i>Salmonella</i> reside within F4/80+ cells the cecum.

<p>C57BL/6 WT (n = 5) (A+C) or Caspase-1−/− (n = 5) (B+D) mice were infected with 1000 cfu of <i>flgM⁻ Salmonella</i> containing a stable GFP expressing plasmid. Intestinal epithelial cells were stained using TROMA (A+B), and phagocytes were stained using F4/80 (C+D) antibody. Red = TROMA-1 (A, B) and F4/80 (C, D); green = GFP (<i>Salmonella</i>). Shown are representative images from 10 mice examined.</p

The innate immune receptor TLR5 is dispensable in the attenuation of <i>flgM⁻</i>.

<p>C57BL/6 WT (n = 14) and TLR5−/− (n = 16) mice were infected with 1000 CFU <i>flgM⁻ Salmonella</i>. Bacterial burden in the cecum (A). Spleen (B). Liver (C). MLN (D). Representative histology of the cecum for WT mice (E) or TLR5−/− (F), and liver of WT mice (G) or TLR5−/− (H). Liver images (200X magnification), cecal images (100X magnification). Figures A–D represent data from three independent experiments. Mann-Whitney test * = p<0.05. ** = p<0.01.(I) Histological scores for changes in the cecal pathology as described in Fig. 4. Figures A–D represent data from three independent experiments. Mann-Whitney test *** = p<0.001.</p

Wildtype <i>Salmonella</i> efficiently evades flagellin detection during acute mucosal infection in streptomycin-pretreated mice.

<p>Bacterial burden of WT C57BL/6 (n = 11) and caspase-1−/− (n = 8) mice infected with 1000 cfu WT SL1344 <i>Salmonella</i> in the cecum (A), spleen (B), liver (C), MLN (D). Bacterial burden of WT C57BL/6 (n = 12) and TLR5−/− (n = 13) mice infected with 1000 cfu wildtype <i>Salmonella</i> in the cecum (E), spleen (F), liver (G), MLN (H). Figures A–D are the combined data from two independent experiments. Figures E–H are the combined data from three independent experiments. Mann-Whitney test. * = p<0.05. ** = p<0.01. *** = p<0.001.</p