Role of macrophages in regression of myocardial fibrosis following alleviation of left ventricular pressure overload.

Sustained hemodynamic pressure overload (PO) produced by murine transverse aortic constriction (TAC) causes myocardial fibrosis; removal of TAC (unTAC) returns left ventricle (LV) hemodynamic load to normal and results in significant, but incomplete regression of myocardial fibrosis. However, the cellular mechanisms that result in these outcomes have not been defined. The objective was to determine temporal changes in myocardial macrophage phenotype in TAC and unTAC and determine whether macrophage depletion alters collagen degradation after unTAC. Myocardial macrophage abundance and phenotype were assessed by immunohistochemistry, flow cytometry, and gene expression by RT-PCR in control (non-TAC), 2 wk, 4 wk TAC, and 2 wk, 4 wk, and 6 wk unTAC. Myocardial cytokine profiles and collagen-degrading enzymes were determined by immunoassay and immunoblots. Initial collagen degradation was detected with collagen-hybridizing peptide (CHP). At unTAC, macrophages were depleted with clodronate liposomes, and endpoints were measured at 2 wk unTAC. Macrophage number had a defined temporal pattern: increased in 2 wk and 4 wk TAC, followed by increases at 2 wk unTAC (over 4 wk TAC) that then decreased at 4 wk and 6 wk unTAC. At 2 wk unTAC, macrophage area was significantly increased and was regionally associated with CHP reactivity. Cytokine profiles in unTAC reflected a proinflammatory milieu versus the TAC-induced profibrotic milieu. Single-cell sequencing analysis of 2 wk TAC versus 2 and 6 wk unTAC revealed distinct macrophage gene expression profiles at each time point demonstrating unique macrophage populations in unTAC versus TAC myocardium. Clodronate liposome depletion at unTAC reduced CHP reactivity and decreased cathepsin K and proMMP2. We conclude that temporal changes in number and phenotype of macrophages play a critical role in both TAC-induced development and unTAC-mediated partial, but incomplete, regression of myocardial fibrosis.NEW & NOTEWORTHY Our novel findings highlight the dynamic changes in myocardial macrophage populations that occur in response to PO and after alleviation of PO. Our data demonstrated, for the first time, a potential benefit of macrophages in contributing to collagen degradation and the partial regression of interstitial fibrosis following normalization of hemodynamic load.

In vitro bioreactor for mechanical control and characterization of tissue constructs.

Cardiac fibrosis is a key contributor to the onset and progression of heart failure and occurs from extracellular matrix accumulation via activated cardiac fibroblasts. Cardiac fibroblasts activate in response to mechanical stress and have been studied in the past by applying forces and deformations to three-dimensional, cell-seeded gels and tissue constructs in vitro. Unfortunately, previous stretching platforms have traditionally not enabled mechanical property assessment to be performed with an efficient throughput, thereby limiting the full potential of in vitro mechanobiology studies. We have developed a novel in vitro platform to study cell-populated tissue constructs under dynamic mechanical stimulation while also performing repeatable, non-destructive stress-strain tests in living constructs. Additionally, this platform can perform these tests across all constructs in a multi-well plate simultaneously, providing exciting potential for direct, functional readouts in future screening applications. In our pilot application, we showed that cyclically stretching cell-populated tissue constructs composed of murine cardiac fibroblasts within a 3D fibrin matrix leads to collagen accumulation and increased tissue stiffness over a three-day time course. Results of this study validate our platform's ability to apply mechanical loads to tissues while performing live mechanical analyses to observe cell-mediated tissue remodeling.

Mechanisms that limit regression of myocardial fibrosis following removal of left ventricular pressure overload.

Left ventricular pressure overload (LVPO) can develop from antecedent diseases such as aortic valve stenosis and systemic hypertension and is characterized by accumulation of myocardial extracellular matrix (ECM). Evidence from patient and animal models supports limited reductions in ECM following alleviation of PO, however, mechanisms that control the extent and timing of ECM regression are undefined. LVPO, induced by 4 wk of transverse aortic constriction (TAC) in mice, was alleviated by removal of the band (unTAC). Cardiomyocyte cross-sectional area, collagen volume fraction (CVF), myocardial stiffness, and collagen degradation were assessed for: control, 2-wk TAC, 4-wk TAC, 4-wk TAC + 2-wk unTAC, 4-wk TAC + 4-wk unTAC, and 4-wk TAC + 6-wk unTAC. When compared with 4-wk TAC, 2-wk unTAC resulted in increased reactivity of collagen hybridizing peptide (CHP) (representing initiation of collagen degradation), increased levels of collagenases and gelatinases, decreased levels of collagen cross-linking enzymes, but no change in CVF. When compared with 2-wk unTAC, 4-wk unTAC demonstrated decreased CVF, which did not decline to control values. At 4-wk and 6-wk unTAC, CHP reactivity and mediators of ECM degradation were reduced versus 2-wk unTAC, whereas levels of tissue inhibitor of metalloproteinase (TIMP)-1 increased. ECM homeostasis changed in a time-dependent manner after removal of LVPO and is characterized by early increases in collagen degradation, followed by a later dampening of this process. Tempered ECM degradation with time is predicted to contribute to the finding that normalization of hemodynamic overload alone does not completely regress myocardial fibrosis.NEW & NOTEWORTHY In this study, a murine model demonstrated persistent interstitial fibrosis and myocardial stiffness following alleviation of pressure overload.

Ensemble machine learning model identifies patients with HFpEF from matrix-related plasma biomarkers.

Arterial hypertension can lead to structural changes within the heart including left ventricular hypertrophy (LVH) and eventually heart failure with preserved ejection fraction (HFpEF). The initial diagnosis of HFpEF is costly and generally based on later stage remodeling; thus, improved predictive diagnostic tools offer potential clinical benefit. Recent work has shown predictive value of multibiomarker plasma panels for the classification of patients with LVH and HFpEF. We hypothesized that machine learning algorithms could substantially improve the predictive value of circulating plasma biomarkers by leveraging more sophisticated statistical approaches. In this work, we developed an ensemble classification algorithm for the diagnosis of HFpEF within a population of 480 individuals including patients with HFpEF, patients with LVH, and referent control patients. Algorithms showed strong diagnostic performance with receiver-operating-characteristic curve (ROC) areas of 0.92 for identifying patients with LVH and 0.90 for identifying patients with HFpEF using demographic information, plasma biomarkers related to extracellular matrix remodeling, and echocardiogram data. More impressively, the ensemble algorithm produced an ROC area of 0.88 for HFpEF diagnosis using only demographic and plasma panel data. Our findings demonstrate that machine learning-based classification algorithms show promise as a noninvasive diagnostic tool for HFpEF, while also suggesting priority biomarkers for future mechanistic studies to elucidate more specific regulatory roles.NEW & NOTEWORTHY Machine learning algorithms correctly classified patients with heart failure with preserved ejection fraction with over 90% area under receiver-operating-characteristic curves. Classifications using multidomain features (demographics and circulating biomarkers and echo-based ventricle metrics) proved more accurate than previous studies using single-domain features alone. Excitingly, HFpEF diagnoses were generally accurate even without echo-based measurements, demonstrating that such algorithms could provide an early screening tool using blood-based measurements before sophisticated imaging.

Phenotypic characterization of primary cardiac fibroblasts from patients with HFpEF.

Heart failure is a leading cause of hospitalizations and mortality worldwide. Heart failure with a preserved ejection fraction (HFpEF) represents a significant clinical challenge due to the lack of available treatment modalities for patients diagnosed with HFpEF. One symptom of HFpEF is impaired diastolic function that is associated with increases in left ventricular stiffness. Increases in myocardial fibrillar collagen content is one factor contributing to increases in myocardial stiffness. Cardiac fibroblasts are the primary cell type that produce fibrillar collagen in the heart. However, relatively little is known regarding phenotypic changes in cardiac fibroblasts in HFpEF myocardium. In the current study, cardiac fibroblasts were established from left ventricular epicardial biopsies obtained from patients undergoing cardiovascular interventions and divided into three categories: Referent control, hypertension without a heart failure designation (HTN (-) HFpEF), and hypertension with heart failure (HTN (+) HFpEF). Biopsies were evaluated for cardiac myocyte cross-sectional area (CSA) and collagen volume fraction. Primary fibroblast cultures were assessed for differences in proliferation and protein expression of collagen I, Membrane Type 1-Matrix Metalloproteinase (MT1-MMP), and α smooth muscle actin (αSMA). Biopsies from HTN (-) HFpEF and HTN (+) HFpEF exhibited increases in myocyte CSA over referent control although only HTN (+) HFpEF exhibited significant increases in fibrillar collagen content. No significant changes in proliferation or αSMA was detected in HTN (-) HFpEF or HTN (+) HFpEF cultures versus referent control. Significant increases in production of collagen I was detected in HF (-) HFpEF fibroblasts, whereas significant decreases in MT1-MMP levels were measured in HTN (+) HFpEF cells. We conclude that epicardial biopsies provide a viable source for primary fibroblast cultures and that phenotypic differences are demonstrated by HTN (-) HFpEF and HTN (+) HFpEF cells versus referent control.

Racial difference in atrial size and extracellular matrix homeostatic response to hypertension: Is this a potential mechanism of reduced atrial fibrillation in African Americans?

BACKGROUND: Atrial fibrillation (AF) is less common in African Americans (AA) than Caucasians (C) despite a higher prevalence of risk factors such as hypertension (HTN). OBJECTIVE: Test the hypothesis that differences in extracellular matrix (ECM) between AA and C in response to HTN might attenuate atrial enlargement and alter myocardial fibrosis. METHODS: ECM-related plasma biomarkers and echo data were collected from 326 C and 129 AA subjects with no history of AF, stratified by the presence of HTN, HTN with left ventricular hypertrophy (LVH), or HTN with LVH and heart failure with preserved ejection fraction (HFpEF). RESULTS: Left atrial size was significantly smaller and the extent of enlargement in the presence of HTN was less in AA despite similar ventricular relative wall thickness, echocardiographic measures of diastolic function, and 6 minute-walk-test. AA had significantly lower levels of collagen I telopeptide and higher levels of collagen I propeptide among all strata, suggesting unique collagen homeostasis. Matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) showed a distinctive response to HTN in AA, with significantly lower levels of MMP-2, MMP-3, and MMP-8 in AA with HTN and significantly lower levels of TIMP-1 and TIMP-3 in AA with HTN and AA with LVH. AA had significantly lower levels of NT-pro-BNP in all strata. CONCLUSION: This cross-sectional study demonstrates a racial disparity in ECM blood biomarkers and atrial remodeling in response to HTN and in the development of LVH and HFpEF that may partly help explain the decreased risk of AF in AA.

Acute Hemodynamic Effects of Cardiac Resynchronization Therapy Versus Alternative Pacing Strategies in Patients With Left Ventricular Assist Devices.

Background The hemodynamic effects of cardiac resynchronization therapy in patients with left ventricular assist devices (LVADs) are uncharacterized. We aimed to quantify the hemodynamic effects of different ventricular pacing configurations in patients with LVADs, focusing on short-term changes in load-independent right ventricular (RV) contractility. Methods and Results Patients with LVADs underwent right heart catheterization during spontaneous respiration without sedation and with pressures recorded at end expiration. Right heart catheterization was performed at different pacemaker configurations (biventricular pacing, left ventricular pacing, RV pacing, and unpaced conduction) in a randomly generated sequence with >3 minutes between configuration change and hemodynamic assessment. The right heart catheterization operator was blinded to the sequence. RV maximal change in pressure over time normalized to instantaneous pressure was calculated from digitized hemodynamic waveforms, consistent with a previously validated protocol. Fifteen patients with LVADs who were in sinus rhythm were included. Load-independent RV contractility, as assessed by RV maximal change in pressure over time normalized to instantaneous pressure, was higher in biventricular pacing compared with unpaced conduction (15.7±7.6 versus 11.0±4.0 s-1; P=0.003). Thermodilution cardiac output was higher in biventricular pacing compared with unpaced conduction (4.48±0.7 versus 4.38±0.8 L/min; P=0.05). There were no significant differences in heart rate, ventricular filling pressures, or atrioventricular valvular regurgitation across all pacing configurations. Conclusions Biventricular pacing acutely improves load-independent RV contractility in patients with LVADs. Even in these patients with mechanical left ventricular unloading via LVAD who were relative pacing nonresponders (required LVAD support despite cardiac resynchronization therapy), biventricular pacing was acutely beneficial to RV contractility.

SPARC production by bone marrow-derived cells contributes to myocardial fibrosis in pressure overload.

In human heart failure and in murine hearts with left-ventricular pressure overload (LVPO), increases in fibrosis are associated with increases in myocardial stiffness. Secreted protein acidic and rich in cysteine (SPARC) is shown to be necessary for both cardiac fibrosis and increases in myocardial stiffness in response to LVPO; however, cellular sources of cardiac SPARC are incompletely defined. Irradiation and bone marrow transfer were undertaken to test the hypothesis that SPARC expression by bone marrow-derived cells is an important mediator of fibrosis in LVPO. In recipient SPARC-null mice transplanted with donor wild-type (WT) bone marrow and subjected to LVPO, levels of fibrosis similar to that of WT mice were found despite the lack of SPARC expression by resident cells. In recipient WT mice with donor SPARC-null bone marrow, significantly less fibrosis versus that of WT mice was found despite the expression of SPARC by resident cells. Increases in myocardial stiffness followed a similar pattern to that of collagen deposition. Myocardial macrophages were significantly reduced in SPARC-null mice with LVPO versus that of WT mice. Recipient SPARC-null mice transplanted with donor WT bone marrow exhibited an increase in cardiac macrophages versus that of SPARC-null LVPO and donor WT mice with recipient SPARC-null bone marrow. Expression of vascular cellular adhesion molecule (VCAM), a previously identified binding partner of SPARC, was assessed in all groups and with the exception of WT mice, increases in VCAM immunoreactivity with LVPO were observed. However, no differences in VCAM expression between bone marrow transplant groups were noted. In conclusion, SPARC expression by bone marrow-derived cells was critical for fibrotic deposition of collagen and influenced the expansion of myocardial macrophages in response to LVPO.NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in LV and myocardial stiffness represent pivotal consequences of chronic pressure overload (PO). In this study, a murine model of cardiac fibrosis induced by PO was used to demonstrate a critical function of SPARC in bone marrow-derived cells that drives cardiac fibrosis and increases in cardiac macrophages.

Prediction of heart failure hospitalizations based on the direct measurement of intrathoracic impedance.

AIMS: OptiVol fluid index was developed as a transthoracic impedance-based indicator of short-term risk for heart failure hospitalization (HFH). OptiVol is calculated as the accumulating difference between daily impedance (measured impedance) and long-term average impedance (reference impedance). Measured impedance alone was thought to have limited prognostic utility; however, measured impedance has the advantage of being simple, direct, and possibly additive to OptiVol fluid index in establishing long-term HFH risk. We tested the hypothesis that directly measured impedance has independent prognostic value in predicting long-term HFH risk and that changes in measured impedance result in a change in predicted long-term HFH risk. METHODS AND RESULTS: A retrospective analysis of 1719 patients studied in PARTNERS-HF, FAST, and RAFT studies was performed. Baseline measured impedance was determined using daily values averaged over 1 month, from Month 6 to 7 post implant; change in measured impedance was determined from values averaged over 1 month, from Month 7 to 8 post implant compared with baseline. The predictive value of baseline measured impedance for HFHs was assessed beginning 7 months post implant. The predictive value of a change in measured impedance for a change in HFHs was assessed beginning 8 months post implant. Baseline measured impedance successfully predicted HFHs. For example, 3 year HFH rate for low baseline impedance < 70 Ω was 23%; for high baseline impedance ≥ 70 Ω was 15% (P < 0.001). Changes in measured impedance resulted in changes in predicted HFHs. For example, when a baseline impedance of ≥70 fell during follow-up to <70 Ω, the subsequent HFHs were 15% compared with 4% in patients with measured impedance that remained >70 Ω (P = 0.004). In addition, when baseline measured impedance fell during follow-up by >1%, 2%, or 3%, subsequent HFHs increased to 13%, 17%, or 18%, respectively. Finally, the prognostic value of measured impedance was additive to the prognostic value of the OptiVol fluid index. CONCLUSIONS: Direct measurements of intrathoracic impedance using an implanted device can be used to stratify patients at varying risk of long-term HFH. These direct measurements of impedance have practical clinical appeal because they are simple, continuous, and ambulatory.

Pressure overload generates a cardiac-specific profile of inflammatory mediators.

Mechanisms that contribute to myocardial fibrosis, particularly in response to left ventricular pressure overload (LVPO), remain poorly defined. To test the hypothesis that a myocardial-specific profile of secreted factors is produced in response to PO, levels of 44 factors implicated in immune cell recruitment and function were assessed in a murine model of cardiac hypertrophy and compared with levels produced in a model of pulmonary fibrosis (PF). Mice subjected to PO were assessed at 1 and 4 wk. Protein from plasma, LV, lungs, and kidneys were analyzed by specific protein array analysis in parallel with protein from mice subjected to silica-instilled PF. Of the 44 factors assessed, 13 proteins were elevated in 1-wk PO myocardium, whereas 18 proteins were found increased in fibrotic lung. Eight of those increased in 1-wk LVPO were not found to be increased in fibrotic lungs (CCL-11, CCL-12, CCL-17, CCL-19, CCL-21, CCL-22, IL-16, and VEGF). Additionally, six factors were increased in plasma of 1-wk LVPO in the absence of increases in myocardial levels. In contrast, in mice with PF, no factors were found increased in plasma that were not elevated in lung tissue. Of those factors increased at 1 wk, only TIMP-1 remained elevated at 4 wk of LVPO. Immunohistochemistry of myocardial vasculature at 1 and 4 wk revealed similar amounts of total vasculature; however, evidence of activated endothelium was observed at 1 wk and, to a lesser extent, at 4 wk LVPO. In conclusion, PO myocardium generated a unique signature of cytokine expression versus that of fibrotic lung.NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in myocardial stiffness represent pivotal consequences of chronic pressure overload (PO). In this study, cytokine profiles produced in a murine model of cardiac fibrosis induced by PO were compared with those produced in response to silica-induced lung fibrosis. A unique profile of cardiac tissue-specific and plasma-derived factors generated in response to PO are reported.

Focusing Heart Failure Research on Myocardial Fibrosis to Prioritize Translation.

Heart failure (HF) has traditionally been defined by symptoms of fluid accumulation and poor perfusion, but it is now recognized that specific HF classifications hold prognostic and therapeutic relevance. Specifically, HF with reduced ejection fraction is characterized by reduced left ventricular systolic pump function and dilation and HF with preserved ejection fraction is characterized primarily by abnormal left ventricular filling (diastolic failure) with relatively preserved left ventricular systolic function. These forms of HF are distributed equally among patients with HF and likely require distinctly different strategies to mitigate the morbidity, mortality, and medical resource utilization of this disease. In particular, HF is a significant medical issue within the US Department of Veterans Affairs (VA) hospital system and constitutes a major translational research priority for the VA. Because a common underpinning of both HF with reduced ejection fraction and HF with preserved ejection fraction seems to be changes in the structure and function of the myocardial extracellular matrix, a conference was convened sponsored by the VA, entitled, "Targeting Myocardial Fibrosis in Heart Failure" to explore the extracellular matrix as a potential therapeutic target and to propose specific research directions. The conference was conceptually framed around the hypothesis that although HF with reduced ejection fraction and HF with preserved ejection fraction clearly have distinct mechanisms, they may share modifiable pathways and biological mediators in common. Inflammation and extracellular matrix were identified as major converging themes. A summary of our discussion on unmet challenges and possible solutions to move the field forward, as well as recommendations for future research opportunities, are provided.

Mitochondrial biogenesis induced by the ß2-adrenergic receptor agonist formoterol accelerates podocyte recovery from glomerular injury.

Podocytes have limited ability to recover from injury. Here, we demonstrate that increased mitochondrial biogenesis, to meet the metabolic and energy demand of a cell, accelerates podocyte recovery from injury. Analysis of events induced during podocyte injury and recovery showed marked upregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a transcriptional co-activator of mitochondrial biogenesis, and key components of the mitochondrial electron transport chain. To evaluate our hypothesis that increasing mitochondrial biogenesis enhanced podocyte recovery from injury, we treated injured podocytes with formoterol, a potent, specific, and long-acting ß2-adrenergic receptor agonist that induces mitochondrial biogenesis in vitro and in vivo. Formoterol increased mitochondrial biogenesis and restored mitochondrial morphology and the injury-induced changes to the organization of the actin cytoskeleton in podocytes. Importantly, ß2-adrenergic receptors were found to be present on podocyte membranes. Their knockdown attenuated formoterol-induced mitochondrial biogenesis. To determine the potential clinical relevance of these findings, mouse models of acute nephrotoxic serum nephritis and chronic (Adriamycin [doxorubicin]) glomerulopathy were used. Mice were treated with formoterol post-injury when glomerular dysfunction was established. Strikingly, formoterol accelerated the recovery of glomerular function by reducing proteinuria and ameliorating kidney pathology. Furthermore, formoterol treatment reduced cellular apoptosis and increased the expression of the mitochondrial biogenesis marker PGC-1α and multiple electron transport chain proteins. Thus, our results support ß2-adrenergic receptors as novel therapeutic targets and formoterol as a therapeutic compound for treating podocytopathies.

Increased macrophage-derived SPARC precedes collagen deposition in myocardial fibrosis.

Myocardial fibrosis and the resultant increases in left ventricular stiffness represent pivotal consequences of chronic pressure overload (PO) that impact both functional capacity and the rates of morbid and mortal events. However, the time course and cellular mechanisms that underlie PO-induced fibrosis have not been completely defined. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that has been shown to be required for insoluble collagen deposition and increased myocardial stiffness in response to PO in mice. As macrophages are associated with increases in fibrillar collagen, the hypothesis that macrophages represent a source of increased SPARC production in the PO myocardium was tested. The time course of changes in the myocardial macrophage population was compared with changes in procollagen type I mRNA, production of SPARC, fibrillar collagen accumulation, and diastolic stiffness. In PO hearts, mRNA encoding collagen type I was increased at 3 days, whereas increases in levels of total collagen protein did not occur until 1 wk and were followed by increases in insoluble collagen at 2 wk. Increases in muscle stiffness were not detected before increases in insoluble collagen content (>1 wk). Significant increases in myocardial macrophages that coincided with increased SPARC were found but did not coincide with increases in mRNA encoding collagen type I. Furthermore, immunohistochemistry and flow cytometry identified macrophages as a cellular source of SPARC. We conclude that myocardial macrophages play an important role in the time-dependent increases in SPARC that enhance postsynthetic collagen processing, insoluble collagen content, and myocardial stiffness and contribute to the development of fibrosis. NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in left ventricular and myocardial stiffness represent pivotal consequences of chronic pressure overload. In this study a murine model of cardiac fibrosis induced by pressure overload was used to establish a time course of collagen expression, collagen deposition, and cardiac macrophage expansion.

Attenuation of accelerated renal cystogenesis in Pkd1 mice by renin-angiotensin system blockade.

The intrarenal renin angiotensin system (RAS) is activated in polycystic kidney disease. We have recently shown in the Pkd1 mouse that Gen 2 antisense oligonucleotide (ASO), which suppresses angiotensinogen (Agt) synthesis, is efficacious in slowing kidney cyst formation compared with lisinopril. The aim of this current study was to determine 1) if unilateral nephrectomy accelerates cystogenesis in Pkd1 mice (as previously shown in cilia knockout mice) and 2) whether Agt ASO can slow the progression in this accelerated cystic mouse model. Adult Pkd1 conditional floxed allele mice expressing cre were administered tamoxifen, resulting in global knockout of Pkd1. Three weeks after tamoxifen injection, mice underwent left unilateral nephrectomy. Mice were then treated with Agt ASO (75 mg/kg per week) or aliskiren (20 mg/kg per day)+Agt ASO or control for 8 wk. Unilateral nephrectomy accelerated kidney cyst formation compared with nonnephrectomized mice. Both Agt ASO and Aliskiren+Agt ASO treatments significantly reduced plasma and urinary Agt levels. Blood pressure was lowest in Aliskiren+Agt ASO mice among all treatment groups, and the control group had the highest blood pressure. All mice developed significant kidney cysts at 8 wk after nephrectomy, but Agt ASO and Aliskiren+Agt ASO groups had fewer kidney cysts than controls. Renal pAkt, pS6 levels, and apoptosis were significantly suppressed in those receiving Agt ASO compared with controls. These results indicate that suppressing Agt using an ASO slowed the progression of accelerated cystic kidney disease induced by unilateral nephrectomy in Pkd1 mice by suppressing intrarenal RAS, mammalian target of rapamycin pathway, and cell proliferation.

Palliative Care for Advanced Heart Failure in a Department of Veterans Affairs Regional Hospice Program: Patient Selection, a Treatment Protocol, and Clinical Course.

BACKGROUND: Palliative care for advanced heart failure (HF) is generally recommended. However, few reports have focused on the particulars of treatment, or the clinical course of HF on a specific treatment regimen. OBJECTIVE: Palliation adequate to allow patients to avoid HF admission and die at home. METHODS: Patients from a veterans administration regional practice with multiple, recent hospital admissions were enrolled in community hospice programs. Treatment of HF with reduced left ventricular ejection fraction (HFrEF) included guidelines-directed medical therapy, digoxin, opioids, and oral bumetanide (with metolazone as needed) rather than intravenous diuretics. Levodopa (l-dopa) was added when conventional therapy failed to control symptoms. HF with preserved EF was also treated with bumetanide and opioids. RESULTS: Thirty male veterans, 23 of them with HFrEF, had 90 HF admissions in the 6 months before enrollment, and 3 HF admissions during follow-up of at least 14 months. Twenty-one patients died, 18 of them at home; 14 died within 5 months, and the rest lived much longer. Failure to improve with initial therapy predicted early death. Results were similar for those with reduced and preserved left ventricular ejection fraction. L-dopa was started in 13 patients and tolerated by 8 patients; functional class improved and B-type natriuretic peptide declined after treatment. CONCLUSIONS: With this treatment protocol, there were few HF admissions and patients were able to die at home. It can be used as a guide to therapy, or as an approach that can be tested with additional study.

Intracardiac Pressures Measured Using an Implantable Hemodynamic Monitor: Relationship to Mortality in Patients With Chronic Heart Failure.

BACKGROUND: The purpose of this analysis was to examine whether implantable hemodynamic monitor-derived baseline estimated pulmonary artery diastolic pressure (ePAD) and change from baseline ePAD were independent predictors of all-cause mortality in patients with chronic heart failure. METHODS AND RESULTS: Retrospective analysis used data from 3 studies (n=790 patients; 216 deaths). Baseline ePAD was related to mortality using a multivariable model including baseline and demographic data. Changes in ePAD defined as change from baseline to 6 months and from baseline to 14 days before death or exit from study were related to subsequent mortality, and analysis was adjusted for baseline ePAD. Area under the pressure versus time curve during 180 days before death or exit from study was related to mortality. Baseline ePAD, independent of other covariates, was a significant predictor of mortality (hazard ratio=1.07; 95% confidence interval=1.05-1.09; P<0.0001). Change in ePAD was an independent predictor of mortality (hazard ratio=1.07; 95% confidence interval=1.05-1.100; P=0.0008). Increased ePAD of 3, 4, or 5 mm Hg from baseline to 6 months was associated with increased mortality risk of 23.8%, 32.9%, or 42.8%. Change in ePAD from baseline to 14 days before death or exit from study was higher in patients who died (3.0±8 versus 1.7±10 mm Hg; P=0.003). Area under the pressure versus time curve in the final 180 days before death or exit from study was higher in patients who died versus those alive at end of study (185±668 versus 17±482 mm Hg.days; P=0.006). CONCLUSIONS: Implantable hemodynamic monitor-derived baseline ePAD and change from baseline ePAD were independent predictors of mortality in chronic heart failure patients.

Increased ADAMTS1 mediates SPARC-dependent collagen deposition in the aging myocardium.

Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein highly expressed during fibrosis. Fibrosis is a prominent component of cardiac aging that reduces myocardial elasticity. Previously, we reported that SPARC deletion attenuated myocardial stiffness and collagen deposition in aged mice. To investigate the mechanisms by which SPARC promotes age-related cardiac fibrosis, we evaluated six groups of mice (n = 5-6/group): young (3-5 mo old), middle-aged (10-12 mo old), and old (18-29 mo old) C57BL/6 wild type (WT) and SPARC-null (Null) mice. Collagen content, determined by picrosirius red staining, increased in an age-dependent manner in WT but not in Null mice. A disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) increased in middle-aged and old WT compared with young, whereas in Null mice only old animals showed increased ADAMTS1 expression. Versican, a substrate of ADAMTS1, decreased with age only in WT. To assess the mechanisms of SPARC-induced collagen deposition, we stimulated cardiac fibroblasts with SPARC. SPARC treatment increased secretion of collagen I and ADAMTS1 (both the 110-kDa latent and 87-kDa active forms) into the conditioned media as well as the cellular expression of transforming growth factor-ß1-induced protein (Tgfbi) and phosphorylated Smad2. An ADAMTS1 blocking antibody suppressed the SPARC-induced collagen I secretion, indicating that SPARC promoted collagen production directly through ADAMTS1 interaction. In conclusion, ADAMTS1 is an important mediator of SPARC-regulated cardiac aging.

Lactosylceramide contributes to mitochondrial dysfunction in diabetes.

Sphingolipids have been implicated as key mediators of cell-stress responses and effectors of mitochondrial function. To investigate potential mechanisms underlying mitochondrial dysfunction, an important contributor to diabetic cardiomyopathy, we examined alterations of cardiac sphingolipid metabolism in a mouse with streptozotocin-induced type 1 diabetes. Diabetes increased expression of desaturase 1, (dihydro)ceramide synthase (CerS)2, serine palmitoyl transferase 1, and the rate of ceramide formation by mitochondria-resident CerSs, indicating an activation of ceramide biosynthesis. However, the lack of an increase in mitochondrial ceramide suggests concomitant upregulation of ceramide-metabolizing pathways. Elevated levels of lactosylceramide, one of the initial products in the formation of glycosphingolipids were accompanied with decreased respiration and calcium retention capacity (CRC) in mitochondria from diabetic heart tissue. In baseline mitochondria, lactosylceramide potently suppressed state 3 respiration and decreased CRC, suggesting lactosylceramide as the primary sphingolipid responsible for mitochondrial defects in diabetic hearts. Moreover, knocking down the neutral ceramidase (NCDase) resulted in an increase in lactosylceramide level, suggesting a crosstalk between glucosylceramide synthase- and NCDase-mediated ceramide utilization pathways. These data suggest the glycosphingolipid pathway of ceramide metabolism as a promising target to correct mitochondrial abnormalities associated with type 1 diabetes.

Plasma Biomarkers Reflecting Profibrotic Processes in Heart Failure With a Preserved Ejection Fraction: Data From the Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction Study.

BACKGROUND: Heart failure with preserved ejection fraction is a clinical syndrome that has been associated with changes in the extracellular matrix. The purpose of this study was to determine whether profibrotic biomarkers accurately reflect the presence and severity of disease and underlying pathophysiology and modify response to therapy in patients with heart failure with preserved ejection fraction. METHODS AND RESULTS: Four biomarkers, soluble form of ST2 (an interleukin-1 receptor family member), galectin-3, matrix metalloproteinase-2, and collagen III N-terminal propeptide were measured in the Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction (PARAMOUNT) trial at baseline, 12 and 36 weeks after randomization to valsartan or LCZ696. We examined the relationship between baseline biomarkers, demographic and echocardiographic characteristics, change in primary (change in N-terminal pro B-type natriuretic peptide) and secondary (change in left atrial volume) end points. The median (interquartile range) value for soluble form of ST2 (33 [24.6-48.1] ng/mL) and galectin 3 (17.8 [14.1-22.8] ng/mL) were higher, and for matrix metalloproteinase-2 (188 [155.5-230.6] ng/mL) lower, than in previously published referent controls; collagen III N-terminal propeptide (5.6 [4.3-6.9] ng/mL) was similar to referent control values. All 4 biomarkers correlated with severity of disease as indicated by N-terminal pro B-type natriuretic peptide, E/E', and left atrial volume. Baseline biomarkers did not modify the response to LCZ696 for lowering N-terminal pro B-type natriuretic peptide; however, left atrial volume reduction varied by baseline level of soluble form of ST2 and galectin 3; patients with values less than the observed median (<33 ng/mL soluble form of ST2 and <17.8 ng/mL galectin 3) had reduction in left atrial volume, those above median did not. Although LCZ696 reduced N-terminal pro B-type natriuretic peptide, levels of the other 4 biomarkers were not affected over time. CONCLUSIONS: In patients with heart failure with preserved ejection fraction, biomarkers that reflect collagen homeostasis correlated with the presence and severity of disease and underlying pathophysiology, and may modify the structural response to treatment. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00887588.