Patrick Most

Cardiac adenoviral S100A1 gene delivery rescues failing myocardium

Cardiac-restricted overexpression of the Ca2+-binding protein S100A1 has been shown to lead to increased myocardial contractile performance in vitro and in vivo. Since decreased cardiac expression of S100A1 is a characteristic of heart failure, we tested the hypothesis that S100A1 gene transfer could restore contractile function of failing myocardium. Adenoviral S100A1 gene delivery normalized S100A1 protein expression in a postinfarction rat heart failure model and reversed contractile dysfunction of failing myocardium in vivo and in vitro. S100A1 gene transfer to failing cardiomyocytes restored diminished intracellular Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load mechanistically due to increased SR Ca2+ uptake and reduced SR Ca2+ leak. Moreover, S100A1 gene transfer decreased elevated intracellular Na+ concentrations to levels detected in nonfailing cardiomyocytes, reversed reactivated fetal gene expression, and restored energy supply in failing cardiomyocytes. Intracoronary adenovirus-mediated S100A1 gene delivery in vivo to the postinfarcted failing rat heart normalized myocardial contractile function and Ca2+ handling, which provided support in a physiological context for results found in myocytes. Thus, the present study demonstrates that restoration of S100A1 protein levels in failing myocardium by gene transfer may be a novel therapeutic strategy for the treatment of heart failure.

G Protein–Coupled Receptor Kinase 2 Ablation in Cardiac Myocytes Before or After Myocardial Infarction Prevents Heart Failure

Myocardial G protein-coupled receptor kinase (GRK)2 is a critical regulator of cardiac &bgr;-adrenergic receptor (&bgr;AR) signaling and cardiac function. Its upregulation in heart failure may further depress cardiac function and contribute to mortality in this syndrome. Preventing GRK2 translocation to activated &bgr;AR with a GRK2-derived peptide that binds G&bgr;&ggr; (&bgr;ARKct) has benefited some models of heart failure, but the precise mechanism is uncertain, because GRK2 is still present and &bgr;ARKct has other potential effects. We generated mice in which cardiac myocyte GRK2 expression was normal during embryonic development but was ablated after birth (&agr;MHC-Cre×GRK2 fl/fl) or only after administration of tamoxifen (&agr;MHC-MerCreMer×GRK2 fl/fl) and examined the consequences of GRK2 ablation before and after surgical coronary artery ligation on cardiac adaptation after myocardial infarction. Absence of GRK2 before coronary artery ligation prevented maladaptive postinfarction remodeling and preserved &bgr;AR responsiveness. Strikingly, GRK2 ablation initiated 10 days after infarction increased survival, enhanced cardiac contractile performance, and halted ventricular remodeling. These results demonstrate a specific causal role for GRK2 in postinfarction cardiac remodeling and heart failure and support therapeutic approaches of targeting GRK2 or restoring &bgr;AR signaling by other means to improve outcomes in heart failure.

Stable Myocardial-Specific AAV6-S100A1 Gene Therapy Results in Chronic Functional Heart Failure Rescue

Background— The incidence of heart failure is ever-growing, and it is urgent to develop improved treatments. An attractive approach is gene therapy; however, the clinical barrier has yet to be broken because of several issues, including the lack of an ideal vector supporting safe and long-term myocardial transgene expression. Methods and Results— Here, we show that the use of a recombinant adeno-associated viral (rAAV6) vector containing a novel cardiac-selective enhancer/promoter element can direct stable cardiac expression of a therapeutic transgene, the calcium (Ca2+)-sensing S100A1, in a rat model of heart failure. The chronic heart failure–rescuing properties of myocardial S100A1 expression, the result of improved sarcoplasmic reticulum Ca2+ handling, included improved contractile function and left ventricular remodeling. Adding to the clinical relevance, long-term S100A1 therapy had unique and additive beneficial effects over &bgr;-adrenergic receptor blockade, a current pharmacological heart failure treatment. Conclusions— These findings demonstrate that stable increased expression of S100A1 in the failing heart can be used for long-term reversal of LV dysfunction and remodeling. Thus, long-term, cardiac-targeted rAAV6-S100A1 gene therapy may be of potential clinical utility in human heart failure.

Cardiac AAV9-S100A1 Gene Therapy Rescues Post-Ischemic Heart Failure in a Preclinical Large Animal Model

A protein that regulates cellular calcium, delivered percutaneously to the heart by gene therapy, improves cardiac function in pigs with heart failure and may also be effective in humans. Paving the Way for a Gene Therapy Trial for Heart Failure Heart failure, also known as congestive heart failure, results when, for any number of reasons, the heart no longer pumps enough blood to keep organs perfused and oxygenated. Common in the Western world, heart failure’s first-line treatment is diuretics, which help to remove the excess fluid that pools in the body during heart failure, and β-adrenergic receptor–blocking drugs to interfere with the deleterious effects of the excess catecholamines that accompany this disease. These treatments are effective but do not restore normal heart function; more than half of patients with heart failure die within 5 years. The authors of Pleger et al. now present evidence that a gene therapy approach to augmenting the failing heart’s damaged ability to handle intracellular calcium produces marked improvements in heart function in pigs with heart failure. These results provide enough evidence, the authors say, to justify a clinical trial to see whether this approach could improve patient’s quality of life and survival when added to current treatments. The investigators injected an adenovirus gene therapy vector (AAV9) carrying the gene for S100A1 into pigs experiencing heart failure, induced by the experimental occlusion of a coronary artery. They chose to deliver S100A1 to the failing heart because this calcium-binding protein becomes depleted as the heart fails and is needed for proper regulation of the calcium dynamics within myocardial cells. Two weeks after infarction, the vector (under control of a cardiac-specific promoter) was delivered to non-infarcted regions of the heart (which would eventually fail without treatment). Twelve weeks later, S100A1 protein expression increased and heart function improved. By several measures, calcium handling within the cardiomyocytes was improved, as were markers of mitochondrial energy production. The authors saw no toxic effects of the therapy and verified the cardiac-specific expression of the vector. Although similar gene therapy tests were shown previously to be effective in mice, a test in a large animal model was especially important to verify that this approach is likely to be safe and effective in patients. The volume of successfully transduced tissue in a pig heart is similar to that required for humans. Unlike the rodent, the pig’s heart rate, sarcomeric proteins, and cardiomyocyte calcium handling are all similar to human, and therapeutic vector delivery through a percutaneous catheter, which will be required in patients, could be mimicked in pigs. If the value of this therapy is confirmed in clinical trials, it would be available for patients already suffering heart failure. It would likely prove most useful as an adjunct therapy to currently existing drugs where it could augment the strength of the heart’s contraction. As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)–S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca2+) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)–S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca2+ cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.

Increased proinflammatory endothelial response to S100A8/A9 after preactivation through advanced glycation end products

SummaryBackgroundAtherosclerosis is an inflammatory disease in which a perpetuated activation of NFkappaB via the RAGE (receptor for advanced glycation end products)-MAPK signalling pathway may play an important pathogenetic role. As recently S100 proteins have been identified as ligands of RAGE, we sought to determine the effects of the proinflammatory heterodimer of S100A8/S100A9 on the RAGE-NFkappaB mediated induction of proinflammatory gene expression.MethodsHuman umbilical vein endothelial cells (HUVEC) were preincubated for 72 h with AGE-albumin or unmodified albumin for control, whereas AGE-albumin induction resulted in an upregulation of RAGE. Following this preactivation, cells were stimulated for 48 h with heterodimeric human recombinant S100A8/S100A9.ResultsHeterodimeric S100A8/S100A9 enhanced secretion of IL-6, ICAM-1, VCAM-1 and MCP1 in AGE-albumin pretreated HUVEC in a dose dependent manner. These effects could not be detected after stimulation with the homodimeric proteins S100A8, S100A9, S100A1 and S100B. The effects of heterodimeric S100A8/S100A9 were reduced by inhibition of the MAP-kinase pathways ERK1/2 and p38 by PD 98059 and SB 203580, respectively.ConclusionThe heterodimeric S100A8/S100A9 might therefore play a hitherto unknown role in triggering atherosclerosis in diabetes and renal failure, pathophysiological entities associated with a high AGE burden. Thus, blocking heterodimeric S100A8/S100A9 might represent a novel therapeutic modality in treating atherosclerosis.

S100A1: a regulator of myocardial contractility.

S100A1, a Ca2+ binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been found to colocalize with the sarcoplasmic reticulum (SR) and the contractile filaments in cardiac tissue. Because S100A1 is known to modulate SR Ca2+ handling in skeletal muscle, we sought to investigate the specific role of S100A1 in the regulation of myocardial contractility. To address this issue, we investigated contractile properties of adult cardiomyocytes as well as of engineered heart tissue after S100A1 adenoviral gene transfer. S100A1 gene transfer resulted in a significant increase of unloaded shortening and isometric contraction in isolated cardiomyocytes and engineered heart tissues, respectively. Analysis of intracellular Ca2+ cycling in S100A1-overexpressing cardiomyocytes revealed a significant increase in cytosolic Ca2+ transients, whereas in functional studies on saponin-permeabilized adult cardiomyocytes, the addition of S100A1 protein significantly enhanced SR Ca2+ uptake. Moreover, in Triton-skinned ventricular trabeculae, S100A1 protein significantly decreased myofibrillar Ca2+ sensitivity ([EC50%]) and Ca2+ cooperativity, whereas maximal isometric force remained unchanged. Our data suggest that S100A1 effects are cAMP independent because cellular cAMP levels and protein kinase A-dependent phosphorylation of phospholamban were not altered, and carbachol failed to suppress S100A1 actions. These results show that S100A1 overexpression enhances cardiac contractile performance and establish the concept of S100A1 as a regulator of myocardial contractility. S100A1 thus improves cardiac contractile performance both by regulating SR Ca2+ handling and myofibrillar Ca2+ responsiveness.

Cardiac S100A1 Protein Levels Determine Contractile Performance and Propensity Toward Heart Failure After Myocardial Infarction

Background— Diminished cardiac S100A1 protein levels are characteristic of ischemic and dilated human cardiomyopathy. Because S100A1 has recently been identified as a Ca2+-dependent inotropic factor in the heart, this study sought to explore the pathophysiological relevance of S100A1 levels in development and progression of postischemic heart failure (HF). Methods and Results— S100A1-transgenic (STG) and S100A1-knockout (SKO) mice were subjected to myocardial infarction (MI) by surgical left anterior descending coronary artery ligation, and survival, cardiac function, and remodeling were compared with nontransgenic littermate control (NLC) and wild-type (WT) animals up to 4 weeks. Although MI size was similar in all groups, infarcted S100A1-deficient hearts (SKO-MI) responded with acute contractile decompensation and accelerated transition to HF, rapid onset of cardiac remodeling with augmented apoptosis, and excessive mortality. NLC/WT-MI mice, displaying a progressive decrease in cardiac S100A1 expression, showed a later onset of cardiac remodeling and progression to HF. Infarcted S100A1-overexpressing hearts (STG-MI), however, showed preserved global contractile performance, abrogated apoptosis, and prevention from cardiac hypertrophy and HF with superior survival compared with NLC/WT-MI and SKO-MI. Both Gq-protein–dependent signaling and protein kinase C activation resulted in decreased cardiac S100A1 mRNA and protein levels, whereas Gs-protein–related signaling exerted opposite effects on cardiac S100A1 abundance. Mechanistically, sarcoplasmic reticulum Ca2+ cycling and &bgr;-adrenergic signaling were severely impaired in SKO-MI myocardium but preserved in STG-MI. Conclusions— Our novel proof-of-concept study provides evidence that downregulation of S100A1 protein critically contributes to contractile dysfunction of the diseased heart, which is potentially responsible for driving the progressive downhill clinical course of patients with HF.

Transgenic Overexpression of the Ca2+-binding Protein S100A1 in the Heart Leads to Increased in Vivo Myocardial Contractile Performance

S100A1, a Ca2+-sensing protein of the EF-hand family, is most highly expressed in myocardial tissue, and cardiac S100A1 overexpression in vitro has been shown to enhance myocyte contractile properties. To study the physiological consequences of S100A1 in vivo, transgenic mice were developed with cardiac-restricted overexpression of S100A1. Characterization of two independent transgenic mouse lines with ∼4-fold overexpression of S100A1 in the myocardium revealed a marked augmentation of in vivo basal cardiac function that remained elevated after β-adrenergic receptor stimulation. Contractile function and Ca2+ handling properties were increased in ventricular cardiomyocytes isolated from S100A1 transgenic mice. Enhanced cellular Ca2+ cycling by S100A1 was associated both with increased sarcoplasmic reticulum Ca2+ content and enhanced sarcoplasmic reticulum Ca2+-induced Ca2+ release, and S100A1 was shown to associate with the cardiac ryanodine receptor. No alterations in β-adrenergic signal transduction or major cardiac Ca2+-cycling proteins occurred, and there were no signs of hypertrophy with chronic cardiac S100A1 overexpression. Our findings suggest that S100A1 plays an important in vivo role in the regulation of cardiac function perhaps through interacting with the ryanodine receptor. Because S100A1 protein expression is down-regulated in heart failure, increasing S100A1 expression in the heart may represent a novel means to augment contractility.

Long-term survival of cancer patients compared to heart failure and stroke: A systematic review

BackgroundCancer, heart failure and stroke are among the most common causes of death worldwide. Investigation of the prognostic impact of each disease is important, especially for a better understanding of competing risks. Aim of this study is to provide an overview of long term survival of cancer, heart failure and stroke patients based on the results of large population- and hospital-based studies.MethodsRecords for our study were identified by searches of Medline via Pubmed. We focused on observed and relative age- and sex-adjusted 5-year survival rates for cancer in general and for the four most common malignancies in developed countries, i.e. lung, breast, prostate and colorectal cancer, as well as for heart failure and stroke.ResultsTwenty studies were identified and included for analysis. Five-year observed survival was about 43% for all cancer entities, 40-68% for stroke and 26-52% for heart failure. Five-year age and sex adjusted relative survival was 50-57% for all cancer entities, about 50% for stroke and about 62% for heart failure. In regard to the four most common malignancies in developed countries 5-year relative survival was 12-18% for lung cancer, 73-89% for breast cancer, 50-99% for prostate cancer and about 43-63% for colorectal cancer. Trend analysis revealed a survival improvement over the last decades.ConclusionsThe results indicate that long term survival and prognosis of cancer is not necessarily worse than that of heart failure and stroke. However, a comparison of the prognostic impact of the different diseases is limited, corroborating the necessity for further systematic investigation of competing risks.

Orai1 and Stim1 regulate normal and hypertrophic growth in cardiomyocytes

Cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signalling pathways dependent on extracellular calcium (Ca(2+)) influx that control normal and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1 and stromal interaction molecule 1 (Stim1) in postnatal cardiomyocyte store operated Ca(2+) entry (SOCE) and impact on normal and hypertrophic postnatal cardiomyocyte growth. Employing a combination of siRNA-mediated gene silencing, cultured neonatal rat ventricular cardiomyocytes together with indirect immunofluorescence, epifluorescent Ca(2+) imaging and site-specific protein phosphorylation and real-time mRNA expression analysis, we show for the first time that both Orai1 and Stim1 are present in cardiomyocytes and required for SOCE due to intracellular Ca(2+) store depletion by thapsigargin. Stim1-KD but not Orai1-KD significantly decreased diastolic Ca(2+) levels and caffeine-releasable Ca(2+) from the sarcoplasmic reticulum (SR). Conversely, Orai1-KD but not Stim1-KD significantly diminished basal NRCM cell size, anp and bnp mRNA levels and activity of the calcineurin (CnA) signalling pathway although diminishing both Orai1 and Stim1 proteins similarly attenuated calmodulin kinase II (CamKII) and ERK1/2 activity under basal conditions. Both Orai1- and Stim1-KD completely abrogated phenylephrine (PE) mediated hypertrophic NRCM growth and enhanced natriuretic factor expression by inhibiting G(q)-protein conveyed activation of the CamKII and ERK1/2 signalling pathway. Interestingly, only Orai1-KD but not Stim1-KD prevented Gq-mediated CaN-dependent prohypertrophic signalling. This study shows for the first time that both Orai1 and Stim1 have a key role in cardiomyocyte SOCE regulating both normal and hypertrophic postnatal cardiac growth in vitro.