Targeting mAKAPß expression as a therapeutic approach for ischemic cardiomyopathy.

Ischemic cardiomyopathy is a leading cause of death and an unmet clinical need. Adeno-associated virus (AAV) gene-based therapies hold great promise for treating and preventing heart failure. Previously we showed that muscle A-kinase Anchoring Protein ß (mAKAPß, AKAP6ß), a scaffold protein that organizes perinuclear signalosomes in the cardiomyocyte, is a critical regulator of pathological cardiac hypertrophy. Here, we show that inhibition of mAKAPß expression in stressed adult cardiomyocytes in vitro was cardioprotective, while conditional cardiomyocyte-specific mAKAP gene deletion in mice prevented pathological cardiac remodeling due to myocardial infarction. We developed a new self-complementary serotype 9 AAV gene therapy vector expressing a short hairpin RNA for mAKAPß under the control of a cardiomyocyte-specific promoter (AAV9sc.shmAKAP). This vector efficiently downregulated mAKAPß expression in the mouse heart in vivo. Expression of the shRNA also inhibited mAKAPß expression in human induced cardiomyocytes in vitro. Following myocardial infarction, systemic administration of AAV9sc.shmAKAP prevented the development of pathological cardiac remodeling and heart failure, providing long-term restoration of left ventricular ejection fraction. Our findings provide proof-of-concept for mAKAPß as a therapeutic target for ischemic cardiomyopathy and support the development of a translational pipeline for AAV9sc.shmAKAP for the treatment of heart failure.

Insulin Growth Factor Phenotypes in Heart Failure With Preserved Ejection Fraction, an INSPIRE Registry and CATHGEN Study.

BACKGROUND: The insulin-like growth factor (IGF) axis emerged as an important pathway in heart failure with preserved ejection (HFpEF). We aimed to identify IGF phenotypes associated with HFpEF in the context of high-dimensional proteomic profiling. METHODS: From the INtermountain Healthcare Biological Samples Collection Project and Investigational REgistry for the On-going Study of Disease Origin, Progression and Treatment (Intermountain INSPIRE Registry), we identified 96 patients with HFpEF and matched controls. We performed targeted proteomics, including IGF-1,2, IGF binding proteins (IGFBP) 1-7 and 111 other proteins (EMD Millipore and ELISA). We used partial least square discriminant analysis (PLS-DA) to identify a set of proteins associated with prevalent HFpEF, pulmonary hypertension and 5-year all-cause mortality. K-mean clustering was used to identify IGF phenotypes. RESULTS: Patients with HFpEF had a high prevalence of systemic hypertension (95%) and coronary artery disease (74%). Using PLS-DA, we identified a set of biomarkers, including IGF1,2 and IGFBP 1,2,7, that provided a strong discrimination of HFpEF, pulmonary hypertension and mortality with an area under the curve of 0.91, 0.77 and 0.83, respectively. Using K mean clustering, we identified 3 IGF phenotypes that were independently associated with all-cause 5-year mortality after adjustment for age, NT-proBNP and kidney disease (P = 0.004). Multivariable analysis validated the prognostic value of IGFBP-1 and 2 in the CATHeterization GENetics (CATHGEN) biorepository. CONCLUSION: IGF phenotypes were associated with pulmonary hypertension and mortality in HFpEF.

Generation of AAVS1 integrated doxycycline-inducible CRISPR-Prime Editor human induced pluripotent stem cell line.

Prime editing uses the Cas9 nickase fused to a reverse transcriptase to copy a DNA sequence into a specific locus from a 'prime editing' guide RNA (pegRNA), eliminating the need for double-stranded DNA breaks and donor DNA templates. To facilitate prime editing in human induced pluripotent stem cells (iPSCs), we integrated a doxycycline-inducible Prime Editor protein (PE2) into the AAVS1 genomic safe harbor locus. Prime editing of iPSCs resulted in precise insertion of three nucleotides in HEK3 locus with high efficiency, demonstrating the utility of this approach. This engineered cell line can be used to edit a single or multiple genomic loci by introducing a target-specific pegRNA for precise and effective genome editing to facilitate disease modeling and functional genetics studies.

Immune biomarkers link air pollution exposure to blood pressure in adolescents.

BACKGROUND: Childhood exposure to air pollution contributes to cardiovascular disease in adulthood. Immune and oxidative stress disturbances might mediate the effects of air pollution on the cardiovascular system, but the underlying mechanisms are poorly understood in adolescents. Therefore, we aimed to identify immune biomarkers linking air pollution exposure and blood pressure levels in adolescents. METHODS: We randomly recruited 100 adolescents (mean age, 16 years) from Fresno, California. Using central-site data, spatial-temporal modeling, and distance weighting exposures to the participant's home, we estimated average pollutant levels [particulate matter (PM), polyaromatic hydrocarbons (PAH), ozone (O3), carbon monoxide (CO) and nitrogen oxides (NOx)]. We collected blood samples and vital signs on health visits. Using proteomic platforms, we quantitated markers of inflammation, oxidative stress, coagulation, and endothelial function. Immune cellular characterization was performed via mass cytometry (CyTOF). We investigated associations between pollutant levels, cytokines, immune cell types, and blood pressure (BP) using partial least squares (PLS) and linear regression, while adjusting for important confounders. RESULTS: Using PLS, biomarkers explaining most of the variance in air pollution exposure included markers of oxidative stress (GDF-15 and myeloperoxidase), acute inflammation (C-reactive protein), hemostasis (ADAMTS, D-dimer) and immune cell types such as monocytes. Most of these biomarkers were independently associated with the air pollution levels in fully adjusted regression models. In CyTOF analyses, monocytes were enriched in participants with the highest versus the lowest PM2.5 exposure. In both PLS and linear regression, diastolic BP was independently associated with PM2.5, NO, NO2, CO and PAH456 pollution levels (P ≤ 0.009). Moreover, monocyte levels were independently related to both air pollution and diastolic BP levels (P ≤ 0.010). In in vitro cell assays, plasma of participants with high PM2.5 exposure induced endothelial dysfunction as evaluated by eNOS and ICAM-1 expression and tube formation. CONCLUSIONS: For the first time in adolescents, we found that ambient air pollution levels were associated with oxidative stress, acute inflammation, altered hemostasis, endothelial dysfunction, monocyte enrichment and diastolic blood pressure. Our findings provide new insights on pollution-related immunological and cardiovascular disturbances and advocate preventative measures of air pollution exposure.

Value of Neutrophil to Lymphocyte Ratio and Its Trajectory in Patients Hospitalized With Acute Heart Failure and Preserved Ejection Fraction.

The neutrophil to lymphocyte ratio (NLR) has been proposed as a simple and routinely obtained marker of inflammation. This study sought to determine whether the NLR on admission as well as NLR trajectory would be complementary to the Get with the Guidelines Heart Failure (GWTG-HF) risk score in patients hospitalized with acute heart failure with preserved ejection fraction (HFpEF).Using the Stanford Translational Research Database, we identified 443 patients between January 2002 and December 2013 hospitalized with acute HFpEF and with complete data of NLR both on admission and at discharge. The primary endpoint was all-cause mortality. Mean age was 77 ±â€¯16 years, 58% were female, with a high prevalence of diabetes mellitus (35.4%), coronary artery disease (58.2%), systemic hypertension (96.6%) and history of atrial fibrillation (57.5%). Over a median follow-up of 2.2 years, 121 (27.3%) patients died. The median NLR on admission was 6.5 (IQR 3.6 - 11.1); a majority of patients decreased their NLR during the course of hospitalization. On multivariable Cox modeling, both NLR on admission (HR 1.18 95% CI (1.00 - .38), p = 0.04) and absolute NLR trajectory (HR 1.26 95% CI (1.10 - 1.45), p = 0.001) were shown to be incremental to GWTG-HF risk score (p < 0.05) for outcome prediction. Adding the NLR or absolute NLR trajectory to the GWTG-HF risk score significantly improved the area under the operator-receiver curve and the reclassification up to 3 years after admission.This simple, readily available marker of inflammation may be useful when stratifying the risk of patients hospitalized with HFpEF.

Right ventricular mitochondrial respiratory function in a piglet model of chronic pulmonary hypertension.

OBJECTIVE: We aimed to assess the mitochondrial respiratory capacities in the right ventricle in the setting of ventricular remodeling induced by pressure overload. METHODS: Chronic thromboembolic pulmonary hypertension was induced in 8 piglets over a 12-week period (chronic thromboembolic pulmonary hypertension model). Right ventricular remodeling, right ventricular function, and mitochondrial respiratory function were assessed at 3, 6, and 12 weeks after induction of pulmonary hypertension and were compared with sham animals (n = 5). Right ventricular cardiomyocytes and mitochondrial structure were studied in transmission electronic microscopy after 12 weeks. RESULTS: As of 3 weeks, chronic pressure overload induced right ventricular dilatation, right ventricular hypertrophy, and right ventricular dysfunction. Maladaptive remodeling in the chronic thromboembolic pulmonary hypertension model was confirmed by the decrease of right ventricular pulmonary artery coupling and right fractional area change. Mitochondrial functional assays in permeabilized right ventricular myocardial fibers revealed that oxidative phosphorylation capacities (complex I, complex II, and IV of the mitochondrial respiratory chain) were degraded. Furthermore, no change in substrate preference of mitochondria was found in the overloaded right ventricle. There was a good correlation between maximal mitochondrial oxygen consumption rate and right ventricular pulmonary artery coupling (Pearson coefficient r = 0.83). Transmission electronic microscopy analysis showed that the composition of cardiomyocytes was no different between the chronic thromboembolic pulmonary hypertension group and the sham group. However, mitochondrial structure anomalies were significantly increased in the chronic thromboembolic pulmonary hypertension group. CONCLUSIONS: Mitochondrial respiratory function impairment is involved early in the development of right ventricular dysfunction in a piglet model of chronic thromboembolic pulmonary hypertension. Underlying mechanisms remain to be elucidated.