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SummaryToxicity by recombinant adeno-associated viruses (rAAV) in clinical gene therapy trials (e.g., by rAAV9-mediated fatal liver failure) significantly impairs translation of preclinical rAAV-based cardiac gene therapies employing these vectors. For rAAV5 - a capsid that has shown long-term safety in clinical trials - our translational study demonstrates effective transduction of the left ventricle (LV) of healthy pigs via catheter-based retrograde intravenous delivery (CRID) by means of luciferase reporter gene biodistribution analyses. Combination of rAAV5 with the cardioprotective human geneS100A1(hS100A1) prevents LV myocardial infarct (MI) enlargement and improves LV systolic contractile performance in a porcine model of post-MI chronic cardiac dysfunction. Use of a cardiac-biased promoter ensured the cardiac-directed expression of the therapeutic human transgene without signs of clinical toxicity. The beneficial effects of rAAV5-hS100A1were linked to an attenuated activity of post-MI inflammatory gene networks and this was further validated in a murine model. These novel data together with proven scalable producibility and low pre-existing immunity against rAAV5 in humans may collectively advance clinical translation of rAAV5-hS100A1as a gene therapy medicinal product (GTMP) for a common cardiovascular disease, such as chronic heart failure (CHF).HighlightsRecent fatal adverse events in recombinant adeno-associated virus (AAV)-based clinical gene therapy trials advise the use of rAAV serotypes with proven long-term clinical safety, such as rAAV5, for the pre-clinical development and clinical translation of rAAV-based cardiac gene therapy medicinal products.In a biodistribution and therapeutic proof-of-concept study in farm pigs, rAAV5 was identified as an effective viral vector for cardiac gene transfer and gene therapy for post-ischemic cardiac dysfunction when applied by a standardized cardiac-targeted catheter-based route of administration with the luciferase reporter and cardioprotective human gene S100A1 (hS100A1), respectively.A systems biology analysis linked the novel finding of mitigated inflammatory and activated cardioprotective gene network activities in rAAV5-hS100A1treated postischemic myocardium with improved study left ventricular ejection fraction and prevention of myocardial infarct extension, respectively, which warrants further mechanistic molecular studies.Since rAAV5 has been recently approved for clinical use in a non-cardiac indication and cardiac-targeted S100A1 gene therapy has been effective in numerous pre-clinical animal models of acute and chronic cardiac dysfunction, our translational data support an expedited developmental path for rAAV5-hS100A1throughout investigational new drug-enabling studies towards a first-in-human clinical trial for post-myocardial infarction heart failure.

BackgroundThe EF-hand Ca2+sensor protein S100A1 has been identified as a molecular regulator and enhancer of cardiac performance. S100A1’s ability to recognize and modulate the activity of targets such as SERCA2a and RyR2 in cardiomyocytes has mostly been ascribed to its hydrophobic C-terminalα-helix (residues 75-94).Objective:We therefore hypothesized that a synthetic peptide consisting of residues 75-94 of S100A1 and an N-terminal solubilization tag (S100A1ct) could mimic the performance enhancing effects of S100A1 and may be suitable as a peptide therapeutic to improve the function of diseased hearts.Methods and Results:Applying an integrative translational research pipeline, ranging from computational molecular modeling to large animal cardiac disease models, we characterize S100A1ct as a cell-penetrating peptide with positive inotropic and antiarrhythmic properties in normal and failing myocardiumin vitroandin vivo. This activity translates into improved contractile performance and survival in pre-clinical heart failure models with reduced ejection fraction after S100A1ct systemic administration. Mechanistically, S100A1ct exerts a fast and sustained dose-dependent enhancement of cardiomyocyte Ca2+cycling and prevents ß-adrenergic receptor triggered Ca2+imbalances by targeting SERCA2a and RyR2 activity. Modeling suggests that S100A1ct may stimulate SERCA2a by interacting with the sarcoplasmic transmembrane segments of the multi-span integral membrane Ca2+pump. Incorporation of a cardiomyocyte targeting peptide tag into S100A1ct (cor-S100A1ct) further enhanced its biological and therapeutic potencyin vitroandin vivo.Conclusion:S100A1ct peptide is a promising lead for the development of a novel peptide-based therapeutic against heart failure with reduced ejection fraction.

S100A1 is a small EF-type Ca2+sensor protein that belongs to the multigenic S100 protein family. It is abundantly expressed in cardiomyocytes (CMs) and has been described as a key regulator of CM performance due to its unique ability to interact with structural contractile proteins, regulators of cardiac Ca2+cycling, and mitochondrial proteins. However, our understanding of the molecular mechanisms regulating S100A1 protein levels is limited. We used the bioinformatics tool GPS-SUMO2.0 to identify a putative SUMO interacting motif (SIM) on S100A1. Consistently, a S100A1:SUMO interaction assay showed a Ca2+-dependent interaction of S100A1 with SUMO proteins. In neonatal rat ventricular myocytes (NRVM) and COS1 cells, S100A1 protein abundance increased in the presence of overexpressed SUMO1 without affecting the S100A1 mRNA transcript. We then generated S100A1 truncation mutants, where the SIM motif was removed by truncation or in which the core residues of the SIM motif (residues 77-79) were deleted or replaced by alanine. In COS1 cells and NRVM, overexpression of these S100A1 mutants led to elevated S100A1 mutant mRNA levels but failed to produce respective protein levels. Protein expression of these mutants could be rescued from degradation by addition of the proteasome inhibitor MG-132. By using an information-driven approach to dock the three-dimensional structures of S100A1 and SUMO, we predict a novel interaction mode between the SIM in S100A1 and SUMO. This study shows an important role of SUMO:SIM-mediated protein:protein interaction in the regulation of post-translational protein stability, and provides mechanistic insights into the indispensability of the core SIM for S100A1 post-translational stability.

Introduction Our clinical research unveiled chronic heart failure with preserved ejection fraction (HFpEF) as a long-term sequel in survivors of severe pediatric burn injury due to a yet unknown molecular pathomechanism. Applying a standardized rat model, we systematically determined the pathophysiological impact of burn injury on long-term cardiac performance to uncover systemic and molecular pathomechanisms that may cause post-burn HFpEF development. Methods Male adolescent SD-rats were subjected to a 60 % total body surface area (TBSA) full-thickness burn- or sham-trauma and subsequently characterized after burn-injury by serial transthoracic echocardiography, bulk myocardial next-generation sequencing and proteomics as well as RT-PCR, immuno-blotting (IB), histology and plasma proteomics for cardiac performance and molecular alterations, respectively, at 3, 7, 30 and 90days. Results In comparison to the sham-group (SG), animals from the burn-group (BG) recapitulated typical post-burn clinical traits, such as significant loss in body weight (BG 27 % less than SG at 30d, p< 0.05) or skeletal muscle wasting (27 % less at 30d, p< 0.05) in accord with elevated molecular atrophy markers. We show post-burn cardiac muscle wasting (BG 22 % less at 30d, p< 0.05) and persistent markers of cardiac dysfunction in accord with significant histological cardiomyocyte hypotrophy (BG -8 % at 30d, p< 0.05) and significantly diminished left ventricular (LV) global longitudinal strain and isovolumic relaxation time in BGs, while LV-EF remained unchanged. Weighted gene network correlation analysis from bulk myocardial NGS and clinical traits related activation of immunological and pro-fibrotic pathways in post-burn injury hearts to cardiac dysfunction in BGs. Subsequent RT-PCR and histology confirmed significant myocardial accumulation of cardio-depressive damage associated molecular patterns (i.e., S100A8 and A9) and infiltration by granulocytes and monocytes as well as significant LV fibrosis. Serial plasma proteomic analysis indicated elevated plasma levels of S100A8 and A9 and other heart failure markers that mirrored similar changes in human post-burn plasma samples. Conclusions Here we report the development of HFpEF as a novel systemic consequence of severe burn injury in a rodent model, which warrants further mechanistic and translational studies. Cardiac inflammation and fibrosis are known to negatively impact cardiac performance and may be mechanistic key findings that will guide further therapeutic studies and subsequent validation of post-burn heart failure biomarkers Applicability of Research to Practice This model is part of a translational and interdisciplinary experimental and clinical effort to inform pathophysiology and mechanistics of long-term heart failure in burn patients. Echocardiographic data and parameters from this model are currently being used to evaluate adult survivors of severe burn injury for signs of HFpEF.

In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood. We found that inhibitory factor 1 of the mitochondrial FoF1-ATP synthase (ATPIF1), a protein known to inhibit ATP hydrolysis by the reverse function of ATP synthase during ischemia, was significantly upregulated in pathological cardiac hypertrophy induced by pressure overload, myocardial infarction, or α-adrenergic stimulation. Chemical cross-linking mass spectrometry analysis of hearts hypertrophied by pressure overload suggested that increased expression of ATPIF1 promoted the formation of FoF1-ATP synthase nonproductive tetramer. Using ATPIF1 gain- and loss-of-function cell models, we demonstrated that stalled electron flow due to impaired ATP synthase activity triggered mitochondrial ROS generation, which stabilized HIF1α, leading to transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the metabolic switch and protected against the pathological remodeling during chronic stress. These results uncover a function of ATPIF1 in nonischemic hearts, which gives FoF1-ATP synthase a critical role in metabolic rewiring during the pathological remodeling of the heart.

Background Reduced fatty acid oxidation (FAO) is a hallmark of metabolic remodeling in heart failure. Enhancing mitochondrial long-chain fatty acid uptake by Acetyl-CoA carboxylase 2 (ACC2) deletion increases FAO and prevents cardiac dysfunction during chronic stresses, but therapeutic efficacy of this approach has not been determined. Methods Male and female ACC2 f/f-MCM (ACC2KO) and their respective littermate controls were subjected to chronic pressure overload by TAC surgery. Tamoxifen injection 3 weeks after TAC induced ACC2 deletion and increased FAO in ACC2KO mice with pathological hypertrophy. Results ACC2 deletion in mice with pre-existing cardiac pathology promoted FAO in female and male hearts, but improved cardiac function only in female mice. In males, pressure overload caused a downregulation in the mitochondrial oxidative function. Stimulating FAO by ACC2 deletion caused unproductive acyl-carnitine accumulation, which failed to improve cardiac energetics. In contrast, mitochondrial oxidative capacity was sustained in female pressure overloaded hearts and ACC2 deletion improved myocardial energetics. Mechanistically, we revealed a sex-dependent regulation of PPARα signaling pathway in heart failure, which accounted for the differential response to ACC2 deletion. Conclusion Metabolic remodeling in the failing heart is sex-dependent which could determine the response to metabolic intervention. The findings suggest that both mitochondrial oxidative capacity and substrate preference should be considered for metabolic therapy of heart failure.

Glucose and branched-chain amino acids (BCAAs) are essential nutrients and key determinants of cell growth and stress responses. High BCAA level inhibits glucose metabolism but reciprocal regulation of BCAA metabolism by glucose has not been demonstrated. Here we show that glucose suppresses BCAA catabolism in cardiomyocytes to promote hypertrophic response. High glucose inhibits CREB stimulated KLF15 transcription resulting in downregulation of enzymes in the BCAA catabolism pathway. Accumulation of BCAA through the glucose-KLF15-BCAA degradation axis is required for the activation of mTOR signaling during the hypertrophic growth of cardiomyocytes. Restoration of KLF15 prevents cardiac hypertrophy in response to pressure overload in wildtype mice but not in mutant mice deficient of BCAA degradation gene. Thus, regulation of KLF15 transcription by glucose is critical for the glucose-BCAA circuit which controls a cascade of obligatory metabolic responses previously unrecognized for cell growth., Hypertrophic cardiomyocytes switch their metabolism from fatty acid oxidation to glucose use, but the functional role of this change is unclear. Here the authors show that high intracellular glucose inhibits the degradation of branched-chain amino acids, which is required for the activation of pro-growth mTOR signaling.

The stress-responsive epigenetic repressor histone deacetylase 4 (HDAC4) regulates cardiac gene expression. Here we show that the levels of an N-terminal proteolytically derived fragment of HDAC4, termed HDAC4-NT, are lower in failing mouse hearts than in healthy control hearts. Virus-mediated transfer of the portion of the Hdac4 gene encoding HDAC4-NT into the mouse myocardium protected the heart from remodeling and failure; this was associated with decreased expression of Nr4a1, which encodes a nuclear orphan receptor, and decreased NR4A1-dependent activation of the hexosamine biosynthetic pathway (HBP). Conversely, exercise enhanced HDAC4-NT levels, and mice with a cardiomyocyte-specific deletion of Hdac4 show reduced exercise capacity, which was characterized by cardiac fatigue and increased expression of Nr4a1. Mechanistically, we found that NR4A1 negatively regulated contractile function in a manner that depended on the HBP and the calcium sensor STIM1. Our work describes a new regulatory axis in which epigenetic regulation of a metabolic pathway affects calcium handling. Activation of this axis during intermittent physiological stress promotes cardiac function, whereas its impairment in sustained pathological cardiac stress leads to heart failure.

Rationale: Hypertrophied hearts switch from mainly using fatty acids (FAs) to an increased reliance on glucose for energy production. It has been shown that preserving FA oxidation (FAO) prevents the pathological shift of substrate preference, preserves cardiac function and energetics, and reduces cardiomyocyte hypertrophy during cardiac stresses. However, it remains elusive whether substrate metabolism regulates cardiomyocyte hypertrophy directly or via a secondary effect of improving cardiac energetics. Objective: The goal of this study was to determine the mechanisms of how preservation of FAO prevents the hypertrophic growth of cardiomyocytes. Methods and Results: We cultured adult rat cardiomyocytes in a medium containing glucose and mixed-chain FAs and induced pathological hypertrophy by phenylephrine. Phenylephrine-induced hypertrophy was associated with increased glucose consumption and higher intracellular aspartate levels, resulting in increased synthesis of nucleotides, RNA, and proteins. These changes could be prevented by increasing FAO via deletion of ACC2 (acetyl-CoA-carboxylase 2) in phenylephrine-stimulated cardiomyocytes and in pressure overload–induced cardiac hypertrophy in vivo. Furthermore, aspartate supplementation was sufficient to reverse the antihypertrophic effect of ACC2 deletion demonstrating a causal role of elevated aspartate level in cardiomyocyte hypertrophy. 15N and 13C stable isotope tracing revealed that glucose but not glutamine contributed to increased biosynthesis of aspartate, which supplied nitrogen for nucleotide synthesis during cardiomyocyte hypertrophy. Conclusions: Our data show that increased glucose consumption is required to support aspartate synthesis that drives the increase of biomass during cardiac hypertrophy. Preservation of FAO prevents the shift of metabolic flux into the anabolic pathway and maintains catabolic metabolism for energy production, thus preventing cardiac hypertrophy and improving myocardial energetics.

In cardiac hypertrophy, the adult heart switches from mainly using fatty acids to an increased reliance on glucose to maintain its energetic demands. We have previously shown that preserving fatty acid oxidation (FAO) by cardiac-specific deletion of Acetyl-CoA Carboxylase 2 (ACC2) prevents the shift of substrate preference towards glucose, preserves cardiac function and reduces cardiomyocyte hypertrophy during chronic pressure overload. To determine whether maintaining FAO specifically prevented cardiomyocyte hypertrophy, we treated adult rat cardiomyocytes (CMs) with and without adenoviral-mediated ACC2 knock-down (KD) with phenylephrine (PE, 10 μM). ACC2 KD effectively prevented CM hypertrophy after PE stimulation compared to control CMs (+9±6% vs. 42±6%) in medium supplemented with fatty acids (FA) (5.5 mM glucose, 0.4 mM mixed long-chain FAs and 0.1 mU/ml insulin). Whereas PE stimulation in control CMs increased glucose uptake (+28±8%), accumulation of glycolytic intermediates and lactate (+52±15%), this was normalized after ACC2 KD. Inhibiting FAO by etomoxir or increasing glucose utilization by dichloroacetate abolished the antihypertrophic effects of ACC2 KD after PE stimulation. However, replacing glucose with pyruvate or propionate restored the anti-hypertrophic effect of ACC2 KD. The expansion of TCA cycle pool caused by PE stimulation was also suppressed by ACC KD. Furthermore, ACC2 KD resulted in decreased intracellular amino acid levels, consistent to what has been seen in ACC2 knock-out hearts in vivo. This was accompanied by reduced glutamine consumption from the media (-60±25%) in ACC2 KD CMs, indicating reduced glutamine reliance. Taken together, our data suggest that ACC2 KD reduces the utilization of glucose and glutamine; both substrates known to be required for cell growth in vitro and in vivo. This further supports the hypothesis that maintaining FAO prevents the metabolic switch and CM hypertrophy.

Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca(2+)- and β-adrenergic receptor-triggered proarrhythmogenic SR Ca(2+) leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca(2+) leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca(2+) leak in HF, combining antiarrhythmic potency with chronic inotropic actions.

We have previously shown that preserving fatty acid oxidation (FAO) by cardiac-specific deletion of Acetyl-CoA Carboxylase 2 (ACC2) prevents the shift of substrate preference towards glucose, reduces cardiomyocyte hypertrophy and preserves cardiac function during chronic pressure overload. To determine whether maintaining FAO specifically prevented cardiomyocyte hypertrophy, we treated adult rat cardiomyocytes (CMs) with and without adenoviral-mediated ACC2 knock-down (KD) with phenylephrine (PE, 10 μM). ACC2 KD effectively prevented CM hypertrophy after PE stimulation compared to control CMs (+9±6% vs. 42±6%) in medium supplemented with fatty acids (FA) (5.5 mM glucose, 0.4 mM mixed long-chain FAs and 0.1 mU/ml insulin). Whereas PE stimulation in control CMs increased glucose uptake (+28±8%) this was normalized after ACC2 KD. Inhibiting FAO by etomoxir or increasing glucose utilization by dichloroacetate abolished the beneficial effects of ACC2 KD after PE stimulation. When cultured in glucose-free medium supplemented with FA, ACC2 KD was incapable of preventing cardiomyocyte hypertrophy. However, replacing glucose with pyruvate or propionate restored the anti-hypertrophic effect of ACC2 KD. To determine the therapeutic effects of increasing FAO in vivo , male mice were subjected to transverse aortic constriction (TAC) and sham surgery. Three weeks after surgery, TAC mice had a significant increase in left ventricular (LV) mass as determined by echocardiography compared to sham operated mice (130 vs. 92 mg). At this time point, cardiac-specific ACC2 deletion (iKO) was induced by tamoxifen (tam) administration. ACC2 protein was effectively deleted in iKO sham and TAC hearts compared to controls (CON) 2 weeks post tam injection. FAO was 2-fold higher in iKO TAC vs. CON TAC hearts as assessed by isolated heart perfusion and 13C NMR spectroscopy. CON and iKO mice will be followed until 12 weeks after TAC to determine cardiac function and assess hypertrophy. Together, these data indicate that increasing FAO via inactivation of ACC2 exerts anti-hypertrophic effect in adult cardiomyocytes. Deleting ACC2 in the hypertrophic heart in vivo can increase FAO and represents a valid target to treat pathological cardiac hypertrophy.

Cardiac metabolism is highly adaptive to changes in fuel availability and the energy demand of the heart. This metabolic flexibility is key for the heart to maintain its output during the development and in response to stress. Alterations in substrate preference have been observed in multiple disease states; a clear understanding of their impact on cardiac function in the long term is critical for the development of metabolic therapies. In addition, the contribution of cellular metabolism to growth, survival, and other signalling pathways through the generation of metabolic intermediates has been increasingly noted, adding another layer of complexity to the impact of metabolism on cardiac function. In a quest to understand the complexity of the cardiac metabolic network, genetic tools have been engaged to manipulate cardiac metabolism in a variety of mouse models. The ability to engineer cardiac metabolism in vivo has provided tremendous insights and brought about conceptual innovations. In this review, we will provide an overview of the cardiac metabolic network and highlight alterations observed during cardiac development and pathological hypertrophy. We will focus on consequences of altered substrate preference on cardiac response to chronic stresses through energy providing and non-energy providing pathways.

Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels. Experimental MI in mice induced comparable S100A1 release. S100A1 internalization was observed in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes. In vitro analyses revealed exclusive S100A1 endocytosis by CFs, followed by Toll-like receptor 4 (TLR4)-dependent activation of MAP kinases and NF-κB. CFs exposed to S100A1 assumed an immunomodulatory and anti-fibrotic phenotype characterized i.e. by enhanced intercellular adhesion molecule-1 (ICAM1) and decreased collagen levels. In mice, intracardiac S100A1 injection recapitulated these transcriptional changes. Moreover, antibody-mediated neutralization of S100A1 enlarged infarct size and worsened left ventricular functional performance post-MI. Our study demonstrates alarmin properties for S100A1 from necrotic cardiomyocytes. However, the potentially beneficial role of extracellular S100A1 in MI-related inflammation and repair warrants further investigation.

Low levels of the molecular inotrope S100A1 are sufficient to rescue post-ischemic heart failure (HF). As a prerequisite to clinical application and to determine the safety of myocardial S100A1 DNA-based therapy, we investigated the effects of high myocardial S100A1 expression levels on the cardiac contractile function and occurrence of arrhythmia in a preclinical large animal HF model. At 2 weeks after myocardial infarction domestic pigs presented significant left ventricular (LV) contractile dysfunction. Retrograde application of AAV6-S100A1 (1.5 × 10(13) tvp) via the anterior cardiac vein (ACV) resulted in high-level myocardial S100A1 protein peak expression of up to 95-fold above control. At 14 weeks, pigs with high-level myocardial S100A1 protein overexpression did not show abnormalities in the electrocardiogram. Electrophysiological right ventricular stimulation ruled out an increased susceptibility to monomorphic ventricular arrhythmia. High-level S100A1 protein overexpression in the LV myocardium resulted in a significant increase in LV ejection fraction (LVEF), albeit to a lesser extent than previously reported with low S100A1 protein overexpression. Cardiac remodeling was, however, equally reversed. High myocardial S100A1 protein overexpression neither increases the occurrence of cardiac arrhythmia nor causes detrimental effects on myocardial contractile function in vivo. In contrast, this study demonstrates a broad therapeutic range of S100A1 gene therapy in post-ischemic HF using a preclinical large animal model.

In cardiac hypertrophy, the adult heart switches from mainly using fatty acids to increased reliance on glucose to maintain its energetic demands. Reducing fatty acid overload and further increasing glucose reliance has been suggested to be beneficial in the diseased state. Recently, however, it has been shown that increasing fatty acid oxidation (FAO) by cardiac-specific deletion of Acetyl-CoA Carboxylase 2 (ACC2) maintains cardiac energetics and prevents cardiac dysfunction as well as cardiomyocyte hypertrophy during chronic pressure overload. However, it remained unclear, how increased FAO specifically prevented cardiomyocyte hypertrophy. Thus, the goal of this study was to determine the impact of ACC2 deletion on cardiomyocyte hypertrophy in vitro . Adenoviral-mediated knock-down (KD) of ACC2 in adult rat ventricular cardiomyocytes (CMs) resulted in a 70% downregulation of ACC2 mRNA. In standard CM medium (medium M199, 5.5mM glucose) ACC2 KD resulted in a similar increase in CM growth after phenylephrine (PE) treatment as control CMs (+39±10% in control vs. 41±16% in ACC2 KD CMs). Supplementation of 0.4 mM mixed long-chain fatty acids (FA) and 0.1 mU/ml insulin had no effect on cardiomyocyte morphology or hypertrophic response after PE treatment (+42±6%). However, ACC2 KD effectively prevented CM hypertrophy after PE stimulation in the presence of FA/insulin (+9±6%). Whereas PE stimulation in control CMs increased glucose uptake (+28±8%) and reduced fatty acid uptake (-25±6%), both were normalized after ACC2 KD. Inhibiting FAO by etomoxir or increasing glucose utilization by dichloroacetate abolished the beneficial effects of ACC2 KD after PE stimulation. When cultured in glucose-free medium supplemented with FA, ACC2 KD was incapable of preventing cardiomyocyte hypertrophy. Together, these data indicate that increased FAO after ACC2 deletion prevents cardiomyocyte hypertrophy by reducing glucose reliance, suggesting that rather increasing than reducing FAO is beneficial in cardiac hypertrophy.

Elevated levels of branched-chain amino acids (BCAAs) have recently been implicated in the development of cardiovascular and metabolic diseases, but the molecular mechanisms are unknown. In a mouse model of impaired BCAA catabolism (knockout [KO]), we found that chronic accumulation of BCAAs suppressed glucose metabolism and sensitized the heart to ischemic injury. High levels of BCAAs selectively disrupted mitochondrial pyruvate utilization through inhibition of pyruvate dehydrogenase complex (PDH) activity. Furthermore, downregulation of the hexosamine biosynthetic pathway in KO hearts decreased protein O-linked N-acetylglucosamine (O-GlcNAc) modification and inactivated PDH, resulting in significant decreases in glucose oxidation. Although the metabolic remodeling in KO did not affect baseline cardiac energetics or function, it rendered the heart vulnerable to ischemia-reperfusion injury. Promoting BCAA catabolism or normalizing glucose utilization by overexpressing GLUT1 in the KO heart rescued the metabolic and functional outcome. These observations revealed a novel role of BCAA catabolism in regulating cardiac metabolism and stress response.

Representing the common endpoint of various cardiovascular disorders, heart failure (HF) shows a dramatically growing prevalence. As currently available therapeutic strategies are not capable of terminating the progress of the disease, HF is still associated with a poor clinical prognosis. Among the underlying molecular mechanisms, the loss of cardiomyocyte Ca(2+) cycling integrity plays a key role in the pathophysiological development and progression of the disease. The cardiomyocyte EF-hand Ca(2+) sensor protein S100A1 emerged as a regulator both of sarcoplasmic reticulum (SR), sarcomere and mitochondrial function implicating a significant role in cardiac physiology and dysfunction. In this review, we aim to recapitulate the translation of S100A1-based investigation from first clinical observations over basic research experiments back to a near-clinical setting on the verge of clinical trials today. We also address needs for further developments towards "second-generation" gene therapy and discuss the therapeutic potential of S100A1 gene therapy for HF as a promising novel strategy for future cardiologists. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy".

ABSTRACT Over the past decade, basic and translational research delivered comprehensive evidence for the relevance of the Ca2+-binding protein S100A1 in cardiovascular diseases. Aberrant expression levels of S100A1 surfaced as molecular key defects, driving the pathogenesis of chronic heart failure, arterial and pulmonary hypertension, peripheral artery disease and disturbed myocardial infarction healing. Loss of intracellular S100A1 renders entire Ca2+-controlled networks dysfunctional, thereby leading to cardiomyocyte failure and endothelial dysfunction. Lack of S100A1 release in ischemic myocardium compromises cardiac fibroblast function, entailing impaired damage healing. This review focuses on molecular pathways and signaling cascades regulated by S100A1 in cardiomyocytes, endothelial cells and cardiac fibroblasts in order to provide an overview of our current mechanistic understanding of S100A1's action in cardiovascular pathophysiology.

S100A1 has emerged as a key factor in the control of cardiomyocyte (CM) contractile performance. Improved sarcoplasmic reticulum (SR) function with enhanced Ca2+ resequestration appears critical for its cAMP-independent inotropic effects but raises concerns about potential diastolic SR Ca2+ leakage that might trigger fatal arrhythmias. Thus, the goal of this study was to determine the impact of S100A1 on ryanodine receptor 2 (RyR2)-mediated SR Ca2+ leakage in vitro and in vivo. S100A1 association with the RyR2 was significantly diminished (-50%) in failing cardiomyocytes and hearts with S100A1 downregulation, as shown by co-immunofluorescence, co-immunoprecipitation and proximity ligation assay. Adenoviral-mediated S100A1 overexpression (3-4 fold vs. GFP-control) in quiescent NCs (normal CMs) and FCs (failing CMs) decreased SR Ca2+-frequency (-50 and −40% respectively) and protected from β-AR-triggered diastolic Ca2+-waves (-62 and −58% respectively) in electrically stimulated (2Hz) CMs as assessed by epifluorescent and confocal Ca2+ imaging. In multicellular rat engineered heart tissue (EHT), S100A1-overexpression (6-8 fold vs. GFP-control) protected from Ca2+-triggered after-contractions (ACs) (-50%) with preserved enhancement of isometric twitch force (TF, +40%) at 2Hz. S100A1-mediated rescue of contractile failure of endothelin-1-treated EHT (-50% decrease in TF) was associated with protection from Ca2+-triggered ACs. In mice with post-ischemic heart failure, AAV9-mediated therapeutic administration of S100A1 enhanced S100A1/RyR2 association and prevented epinephrine-induced VTs (70% in MI group vs. 30% in MI-S100A1 group). Mechanistically, S100A1-overexpression changed neither PKA/CaMKII RyR2 phosphorylation pattern nor binding of accessory proteins like FKBP12.6, calmodulin or sorcin to RyR2 but enhanced S100A1/RyR2 stoichiometry. Our data provide evidence that S100A1 interaction with the RyR2 can beneficially modulate and reverse diastolic RyR2 function dysfunction. S100A1 appears to convey a rather unique molecular profile combining cAMP-independent inotropy with protection against Ca2+-triggered arrhythmias.

Gene therapy, aimed at the correction of key pathologies being out of reach for conventional drugs, bears the potential to alter the treatment of cardiovascular diseases radically and thereby of heart failure. Heart failure gene therapy refers to a therapeutic system of targeted drug delivery to the heart that uses formulations of DNA and RNA, whose products determine the therapeutic classification through their biological actions. Among resident cardiac cells, cardiomyocytes have been the therapeutic target of numerous attempts to regenerate systolic and diastolic performance, to reverse remodeling and restore electric stability and metabolism. Although the concept to intervene directly within the genetic and molecular foundation of cardiac cells is simple and elegant, the path to clinical reality has been arduous because of the challenge on delivery technologies and vectors, expression regulation, and complex mechanisms of action of therapeutic gene products. Nonetheless, since the first demonstration of in vivo gene transfer into myocardium, there have been a series of advancements that have driven the evolution of heart failure gene therapy from an experimental tool to the threshold of becoming a viable clinical option. The objective of this review is to discuss the current state of the art in the field and point out inevitable innovations on which the future evolution of heart failure gene therapy into an effective and safe clinical treatment relies.

Rationale: Mice lacking the EF-hand Ca 2+ sensor S100A1 display endothelial dysfunction because of distorted Ca 2+ -activated nitric oxide (NO) generation. Objective: To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization. Methods and Results: Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue. Ensuing studies in S100A1 knockout (SKO) mice subjected to femoral artery resection unveiled insufficient perfusion recovery and high rates of autoamputation. Defective in vivo angiogenesis prompted cellular studies in SKO ECs and human ECs, with small interfering RNA–mediated S100A1 knockdown demonstrating impaired in vitro and in vivo proangiogenic properties (proliferation, migration, tube formation) and attenuated vascular endothelial growth factor (VEGF)–stimulated and hypoxia-stimulated endothelial NO synthase (eNOS) activity. Mechanistically, S100A1 deficiency compromised eNOS activity in ECs by interrupted stimulatory S100A1/eNOS interaction and protein kinase C hyperactivation that resulted in inhibitory eNOS phosphorylation and enhanced VEGF receptor-2 degradation with attenuated VEGF signaling. Ischemic SKO tissue recapitulated the same molecular abnormalities with insufficient in vivo NO generation. Unresolved ischemia entailed excessive VEGF accumulation in SKO mice with aggravated VEGF receptor-2 degradation and blunted in vivo signaling through the proangiogenic phosphoinositide-3-kinase/Akt/eNOS cascade. The NO supplementation strategies rescued defective angiogenesis and salvaged limbs in SKO mice after femoral artery resection. Conclusions: Our study shows for the first time downregulation of S100A1 expression in patients with critical limb ischemia and identifies S100A1 as critical for EC function in postnatal ischemic angiogenesis. These findings link its pathological plasticity in critical limb ischemia to impaired neovascularization, prompting further studies to probe the microvascular therapeutic potential of S100A1.

Heart failure (HF) is the common endpoint of many cardiovascular diseases with a 1-year survival rate of about 50% in advanced stages. Despite increasing survival rates in the past years, current standard therapeutic strategies are far away from being optimal. For this reason, the concept of cardiac gene therapy for the treatment of HF holds great potential to improve disease progression, as it specifically targets key pathologies of diseased cardiomyocytes (CM). The small calcium (Ca(2+))-binding protein S100A1 presents a promising target for cardiac gene therapy, as it has been identified as a central regulator of cardiac performance and the Ca(2+)-driven network within CM. S100A1 was shown to regulate sarcoplasmic reticulum, sarcomere and mitochondrial function by modulating target protein activity. Furthermore, deranged S100A1 expression has been linked to HF in human ischemic and dilated cardiomyopathies as well as in various HF animal models. Proof-of-concept studies in small and large animal models as wells as in human failing CM could demonstrate feasibility and efficacy of S100A1 genetically targeted therapy. This review summarizes the developmental steps of S100A1 gene therapy for the implementation into first human clinical trials.

Heart failure (HF) is the common end point of cardiac diseases. Despite the optimization of therapeutic strategies and the consequent overall reduction in HF-related mortality, the key underlying intracellular signal transduction abnormalities have not been addressed directly. In this regard, the gaps in modern HF therapy include derangement of β-adrenergic receptor (β-AR) signaling, Ca(2+) disbalances, cardiac myocyte death, diastolic dysfunction, and monogenetic cardiomyopathies. In this review we discuss the potential of gene therapy to fill these gaps and rectify abnormalities in intracellular signaling. We also examine current vector technology and currently available vector-delivery strategies, and we delineate promising gene therapy structures. Finally, we analyze potential limitations related to the transfer of successful preclinical gene therapy approaches to HF treatment in the clinic, as well as impending strategies aimed at overcoming these limitations.

Cardiovascular disease is the leading cause of death worldwide, showing a dramatically growing prevalence. It is still associated with a poor clinical prognosis, indicating insufficient long-term treatment success of currently available therapeutic strategies. Investigations of the pathomechanisms underlying cardiovascular disorders uncovered the Ca(2+) binding protein S100A1 as a critical regulator of both cardiac performance and vascular biology. In cardiomyocytes, S100A1 was found to interact with both the sarcoplasmic reticulum ATPase (SERCA2a) and the ryanodine receptor 2 (RyR2), resulting in substantially improved Ca(2+) handling and contractile performance. Additionally, S100A1 has been described to target the cardiac sarcomere and mitochondria, leading to reduced pre-contractile passive tension as well as enhanced oxidative energy generation. In endothelial cells, molecular analyses revealed a stimulatory effect of S100A1 on endothelial NO production by increasing endothelial nitric oxide synthase activity. Emphasizing the pathophysiological relevance of S100A1, myocardial infarction in S100A1 knockout mice resulted in accelerated transition towards heart failure and excessive mortality in comparison with wild-type controls. Mice lacking S100A1 furthermore displayed significantly elevated blood pressure values with abrogated responsiveness to bradykinin. On the other hand, numerous studies in small and large animal heart failure models showed that S100A1 overexpression results in reversed maladaptive myocardial remodeling, long-term rescue of contractile performance, and superior survival in response to myocardial infarction, indicating the potential of S100A1-based therapeutic interventions. In summary, elaborate basic and translational research established S100A1 as a multifaceted therapeutic target in cardiovascular disease, providing a promising novel therapeutic strategy to future cardiologists.