Failure to repair endogenous DNA damage in ß-cells causes adult-onset diabetes in mice.

Age is the greatest risk factor for the development of type 2 diabetes mellitus (T2DM). Age-related decline in organ function is attributed to the accumulation of stochastic damage, including damage to the nuclear genome. Islets of T2DM patients display increased levels of DNA damage. However, whether this is a cause or consequence of the disease has not been elucidated. Here, we asked if spontaneous, endogenous DNA damage in ß-cells can drive ß-cell dysfunction and diabetes, via deletion of Ercc1, a key DNA repair gene, in ß-cells. Mice harboring Ercc1-deficient ß-cells developed adult-onset diabetes as demonstrated by increased random and fasted blood glucose levels, impaired glucose tolerance, and reduced insulin secretion. The inability to repair endogenous DNA damage led to an increase in oxidative DNA damage and apoptosis in ß-cells and a significant loss of ß-cell mass. Using electron microscopy, we identified ß-cells in clear distress that showed an increased cell size, enlarged nuclear size, reduced number of mature insulin granules, and decreased number of mitochondria. Some ß-cells were more affected than others consistent with the stochastic nature of spontaneous DNA damage. Ercc1-deficiency in ß-cells also resulted in loss of ß-cell function as glucose-stimulated insulin secretion and mitochondrial function were impaired in islets isolated from mice harboring Ercc1-deficient ß-cells. These data reveal that unrepaired endogenous DNA damage is sufficient to drive ß-cell dysfunction and provide a mechanism by which age increases the risk of T2DM.

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy.

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1-/D mice). Ckmm-Cre+/- ;Ercc1-/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre+/- ;Ercc1-/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre+/- ;Ercc1-/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre+/- ;Ercc1-/fl and Ercc1-/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.

Novel small molecule inhibition of IKK/NF-κB activation reduces markers of senescence and improves healthspan in mouse models of aging.

Constitutive NF-κB activation is associated with cellular senescence and stem cell dysfunction and rare variants in NF-κB family members are enriched in centenarians. We recently identified a novel small molecule (SR12343) that inhibits IKK/NF-κB activation by disrupting the association between IKKß and NEMO. Here we investigated the therapeutic effects of SR12343 on senescence and aging in three different mouse models. SR12343 reduced senescence-associated beta-galactosidase (SA-ß-gal) activity in oxidative stress-induced senescent mouse embryonic fibroblasts as well as in etoposide-induced senescent human IMR90 cells. Chronic administration of SR12343 to the Ercc1-/∆ and Zmpste24-/- mouse models of accelerated aging reduced markers of cellular senescence and SASP and improved multiple parameters of aging. SR12343 also reduced markers of senescence and increased muscle fiber size in 2-year-old WT mice. Taken together, these results demonstrate that IKK/NF-κB signaling pathway represents a promising target for reducing markers of cellular senescence, extending healthspan and treating age-related diseases.

Senolytics reduce coronavirus-related mortality in old mice.

The COVID-19 pandemic has revealed the pronounced vulnerability of the elderly and chronically ill to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced morbidity and mortality. Cellular senescence contributes to inflammation, multiple chronic diseases, and age-related dysfunction, but effects on responses to viral infection are unclear. Here, we demonstrate that senescent cells (SnCs) become hyper-inflammatory in response to pathogen-associated molecular patterns (PAMPs), including SARS-CoV-2 spike protein-1, increasing expression of viral entry proteins and reducing antiviral gene expression in non-SnCs through a paracrine mechanism. Old mice acutely infected with pathogens that included a SARS-CoV-2-related mouse ß-coronavirus experienced increased senescence and inflammation, with nearly 100% mortality. Targeting SnCs by using senolytic drugs before or after pathogen exposure significantly reduced mortality, cellular senescence, and inflammatory markers and increased antiviral antibodies. Thus, reducing the SnC burden in diseased or aged individuals should enhance resilience and reduce mortality after viral infection, including that of SARS-CoV-2.

An aged immune system drives senescence and ageing of solid organs.

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly1,2. To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein3,4, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence5-7 in the immune system only. We show that Vav-iCre+/-;Ercc1-/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice8-10. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre+/-;Ercc1-/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function11,12. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.

ATM is a key driver of NF-κB-dependent DNA-damage-induced senescence, stem cell dysfunction and aging.

NF-κB is a transcription factor activated in response to inflammatory, genotoxic and oxidative stress and important for driving senescence and aging. Ataxia-telangiectasia mutated (ATM) kinase, a core component of DNA damage response signaling, activates NF-κB in response to genotoxic and oxidative stress via post-translational modifications. Here we demonstrate that ATM is activated in senescent cells in culture and murine tissues from Ercc1-deficient mouse models of accelerated aging, as well as naturally aged mice. Genetic and pharmacologic inhibition of ATM reduced activation of NF-κB and markers of senescence and the senescence-associated secretory phenotype (SASP) in senescent Ercc1-/- MEFs. Ercc1-/Δ mice heterozygous for Atm have reduced NF-κB activity and cellular senescence, improved function of muscle-derived stem/progenetor cells (MDSPCs) and extended healthspan with reduced age-related pathology especially age-related bone and intervertebral disc pathologies. In addition, treatment of Ercc1-/∆ mice with the ATM inhibitor KU-55933 suppressed markers of senescence and SASP. Taken together, these results demonstrate that the ATM kinase is a major mediator of DNA damage-induced, NF-κB-mediated cellular senescence, stem cell dysfunction and aging and thus represents a therapeutic target to slow the progression of aging.

Urinary Extracellular Vesicles Carrying Klotho Improve the Recovery of Renal Function in an Acute Tubular Injury Model.

Acute kidney injury, defined by a rapid deterioration of renal function, is a common complication in hospitalized patients. Among the recent therapeutic options, the use of extracellular vesicles (EVs) is considered a promising strategy. Here we propose a possible therapeutic use of renal-derived EVs isolated from normal urine (urine-derived EVs [uEVs]) in a murine model of acute injury generated by glycerol injection. uEVs accelerated renal recovery, stimulating tubular cell proliferation, reducing the expression of inflammatory and injury markers, and restoring endogenous Klotho loss. When intravenously injected, labeled uEVs localized within injured kidneys and transferred their microRNA cargo. Moreover, uEVs contained the reno-protective Klotho molecule. Murine uEVs derived from Klotho null mice lost the reno-protective effect observed using murine EVs from wild-type mice. This was regained when Klotho-negative murine uEVs were reconstituted with recombinant Klotho. Similarly, ineffective fibroblast EVs acquired reno-protection when engineered with human recombinant Klotho. Our results reveal a novel potential use of uEVs as a new therapeutic strategy for acute kidney injury, highlighting the presence and role of the reno-protective factor Klotho.

Increased Plasma-Free Hemoglobin Levels Identify Hemolysis in Patients With Cardiogenic Shock and a Trans valvular Micro-Axial Flow Pump.

Hemolysis is a potential limitation of percutaneously delivered left-sided mechanical circulatory support pumps, including trans valvular micro-axial flow pumps (TVP). Hemolytic biomarkers among durable left ventricular assist devices include lactate dehydrogenase (LDH) >2.5 times the upper limit of normal (ULN) and plasma-free hemoglobin (pf-Hb) >20 mg/dL. We examined the predictive value of these markers among patients with cardiogenic shock (CS) receiving a TVP. We retrospectively studied records of 116 consecutive patients receiving an Impella TVP at our institution between 2012 and 2017 for CS. Twenty-three met inclusion/exclusion criteria, and had sufficient pf-Hb data for analysis. Area under receiver-operator characteristic (ROC) curve for diagnosing hemolysis were calculated. Mean age was 62 ± 14 years and ejection fraction was 15 ± 5%. Mean duration of support was 5.4 ± 3.5 days. Pre-device LDH levels were >2.5x ULN in 71% (n = 5/7) of 5.0 and 29% of CP patients, while pre-device pf-Hb levels were >20 mg/dL in 14% (n = 1/7) of 5.0 and 25% (n = 4/16) of CP patients. Given elevated baseline LDH and pf-Hb levels, we defined hemolysis as a pf-Hb level >40 mg/dL within 72 h post-implant plus clinical evidence of device-related hemolysis. We identified that 30% (n = 7/23) had device-related hemolysis. Using ROC curve-derived cut-points, an increase in delta pf-Hb by >27mg/dL, not delta LDH, within 24 h after TVP implant (delta pf-Hb: C-statistic = 0.79, sensitivity: 57%, specificity: 93%, p <0.05) was highly predictive of hemolysis. In conclusion, we identified a change in pf-Hb, not LDH, levels is highly sensitive and specific for hemolysis in patients treated with a TVP for CS.

Use of a percutaneous temporary circulatory support device as a bridge to decision during acute decompensation of advanced heart failure.

BACKGROUND: Prognosis is poor for patients with decompensated advanced heart failure (HF) refractory to medical therapy. Evaluating candidacy for durable mechanical circulatory support (MCS), cardiac transplantation, or palliative care is complex, and time is often needed to stabilize the patient hemodynamically. The Impella 5.0 (Abiomed, Danvers, MA) is a minimally invasive axial-flow catheter capable of providing full temporary hemodynamic support. We report a multicenter series on the use of this device for bridge to decision (BTD) in decompensated advanced HF patients. METHODS: In a retrospective evaluation at 3 centers of patients with advanced HF who acutely decompensated and received the Impella 5.0 for BTD, we analyzed demographics, procedural characteristics, in-hospital and intermediate-term outcomes, and in-hospital complications. RESULTS: There were 58 patients who met inclusion criteria from 2010 to 2015. All were inotrope dependent. The mean ejection fraction was 13%, and median age was 59 years (interquartile range, 48-64 years). Mean duration of support was 7 days (range, 0-22 days). Thirty-nine patients survived to next therapy (67%), with most receiving durable MCS (n = 20) or heart transplantation (n = 15). In-hospital complications included bleeding (n = 9) and hemolysis (n = 4). Of patients who survived to the next therapy, 1-year survival was 65% for those who received durable MCS, 87% for those who received a transplant, and 75% for those who were stabilized and weaned. CONCLUSIONS: The Impella 5.0 may provide a BTD strategy for patients with advanced HF and acute hemodynamic instability. Prospective studies are needed to evaluate the safety and effectiveness of this device in this patient population.

Acute Biventricular Mechanical Circulatory Support for Cardiogenic Shock.

BACKGROUND: Biventricular failure is associated with high in-hospital mortality. Limited data regarding the efficacy of biventricular Impella axial flow catheters (BiPella) support for biventricular failure exist. The aim of this study was to explore the clinical utility of percutaneously delivered BiPella as a novel acute mechanical support strategy for patients with cardiogenic shock complicated by biventricular failure. METHODS AND RESULTS: We retrospectively analyzed data from 20 patients receiving BiPella for biventricular failure from 5 tertiary-care hospitals in the United States. Left ventricular support was achieved with an Impella 5.0 (n=8), Impella CP (n=11), or Impella 2.5 (n=1). All patients received the Impella RP for right ventricular (RV) support. BiPella use was recorded in the setting of acute myocardial infarction (n=11), advanced heart failure (n=7), and myocarditis (n=2). Mean flows achieved were 3.4±1.2 and 3.5±0.5 for left ventricular and RV devices, respectively. Total in-hospital mortality was 50%. No intraprocedural mortality was observed. Major complications included limb ischemia (n=1), hemolysis (n=6), and Thrombolysis in Myocardial Infarction major bleeding (n=7). Compared with nonsurvivors, survivors were younger, had a lower number of inotropes or vasopressors used before BiPella, and were more likely to have both devices implanted simultaneously during the same procedure. Compared with nonsurvivors, survivors had lower pulmonary artery pressures and RV stroke work index before BiPella. Indices of RV afterload were quantified for 14 subjects. Among these patients, nonsurvivors had higher pulmonary vascular resistance (6.8; 95% confidence interval [95% CI], 5.5-8.1 versus 1.9; 95% CI, 0.8-3.0; P<0.01), effective pulmonary artery elastance (1129; 95% CI, 876-1383 versus 458; 95% CI, 263-653; P<0.01), and lower pulmonary artery compliance (1.5; 95% CI, 0.9-2.1 versus 2.7; 95% CI, 1.8-3.6; P<0.05). CONCLUSIONS: This is the largest, retrospective analysis of BiPella for cardiogenic shock. BiPella is feasible, reduces cardiac filling pressures and improves cardiac output across a range of causes for cardiogenic shock. Simultaneous left ventricular and RV device implantation and lower RV afterload may be associated with better outcomes with BiPella. Future prospective studies of BiPella for cardiogenic shock are required.

Acute Hemodynamic Effects of Intra-aortic Balloon Counterpulsation Pumps in Advanced Heart Failure.

BACKGROUND: The utility of intra-aortic balloon counterpulsation pumps (IABPs) in low cardiac output states is unknown and no studies have explored the impact of IABP therapy on ventricular workload in patients with advanced heart failure (HF). For these reasons, we explored the acute hemodynamic effects of IABP therapy in patients with advanced HF. METHODS: We prospectively studied 10 consecutive patients with stage D HF referred for IABP placement before left ventricular assist device (LVAD) surgery and compared with 5 control patients with preserved left ventricular (LV) ejection fraction (EF) who did not receive IABP therapy. Hemodynamics were recorded using LV conductance and pulmonary artery catheters. Cardiac index (CI)-responder and CI-nonresponder status was assigned a priori as being "equal to or above" or below the median of the IABP effect on CI, respectively, within 24 hours after IABP activation. RESULTS: Compared with controls, patients with advanced HF had lower LVEF, lower LV end-systolic pressure, lower LV stroke work, and higher LV end-diastolic pressures and volumes before IABP activation. IABP activation reduced LV stroke work primarily by reducing end-systolic pressure. IABP therapy increased CI by a median of 20% as well as increased diastolic pressure time index and the myocardial oxygen supply:demand ratio. Compared with CI-nonresponders, CI-responders had higher systemic vascular resistance, lower right heart filling pressures, and a trend toward lower left heart filling pressures with improved indices of right heart function. Compared with CI-nonresponders, the diastolic pressure time index was increased among CI-responders. CONCLUSIONS: IABP therapy may be effective at reducing LV stroke work, increasing CI, and favorably altering the myocardial oxygen supply:demand ratio in patients with advanced HF, especially among patients with low right heart filling pressures and high systemic vascular resistance.