Understanding Key Mechanisms of Exercise-Induced Cardiac Protection to Mitigate Disease: Current Knowledge and Emerging Concepts.

The benefits of exercise on the heart are well recognized, and clinical studies have demonstrated that exercise is an intervention that can improve cardiac function in heart failure patients. This has led to significant research into understanding the key mechanisms responsible for exercise-induced cardiac protection. Here, we summarize molecular mechanisms that regulate exercise-induced cardiac myocyte growth and proliferation. We discuss in detail the effects of exercise on other cardiac cells, organelles, and systems that have received less or little attention and require further investigation. This includes cardiac excitation and contraction, mitochondrial adaptations, cellular stress responses to promote survival (heat shock response, ubiquitin-proteasome system, autophagy-lysosomal system, endoplasmic reticulum unfolded protein response, DNA damage response), extracellular matrix, inflammatory response, and organ-to-organ crosstalk. We summarize therapeutic strategies targeting known regulators of exercise-induced protection and the challenges translating findings from bench to bedside. We conclude that technological advancements that allow for in-depth profiling of the genome, transcriptome, proteome and metabolome, combined with animal and human studies, provide new opportunities for comprehensively defining the signaling and regulatory aspects of cell/organelle functions that underpin the protective properties of exercise. This is likely to lead to the identification of novel biomarkers and therapeutic targets for heart disease.

Cardiovascular magnetic resonance imaging for sequential assessment of cardiac fibrosis in mice: technical advancements and reverse translation.

Cardiovascular magnetic resonance (CMR) imaging has become an essential technique for the assessment of cardiac function and morphology, and is now routinely used to monitor disease progression and intervention efficacy in the clinic. Cardiac fibrosis is a common characteristic of numerous cardiovascular diseases and often precedes cardiac dysfunction and heart failure. Hence, the detection of cardiac fibrosis is important for both early diagnosis and the provision of guidance for interventions/therapies. Experimental mouse models with genetically and/or surgically induced disease have been widely used to understand mechanisms underlying cardiac fibrosis and to assess new treatment strategies. Improving the appropriate applications of CMR to mouse studies of cardiac fibrosis has the potential to generate new knowledge, and more accurately examine the safety and efficacy of antifibrotic therapies. In this review, we provide 1) a brief overview of different types of cardiac fibrosis, 2) general background on magnetic resonance imaging (MRI), 3) a summary of different CMR techniques used in mice for the assessment of cardiac fibrosis including experimental and technical considerations (contrast agents and pulse sequences), and 4) provide an overview of mouse studies that have serially monitored cardiac fibrosis during disease progression and/or therapeutic interventions. Clinically established CMR protocols have advanced mouse CMR for the detection of cardiac fibrosis, and there is hope that discovery studies in mice will identify new antifibrotic therapies for patients, highlighting the value of both reverse translation and bench-to-bedside research.

Fine-Tuning Cardiac Insulin-Like Growth Factor 1 Receptor Signaling to Promote Health and Longevity.

BACKGROUND: The insulin-like growth factor 1 (IGF1) pathway is a key regulator of cellular metabolism and aging. Although its inhibition promotes longevity across species, the effect of attenuated IGF1 signaling on cardiac aging remains controversial. METHODS: We performed a lifelong study to assess cardiac health and lifespan in 2 cardiomyocyte-specific transgenic mouse models with enhanced versus reduced IGF1 receptor (IGF1R) signaling. Male mice with human IGF1R overexpression or dominant negative phosphoinositide 3-kinase mutation were examined at different life stages by echocardiography, invasive hemodynamics, and treadmill coupled to indirect calorimetry. In vitro assays included cardiac histology, mitochondrial respiration, ATP synthesis, autophagic flux, and targeted metabolome profiling, and immunoblots of key IGF1R downstream targets in mouse and human explanted failing and nonfailing hearts, as well. RESULTS: Young mice with increased IGF1R signaling exhibited superior cardiac function that progressively declined with aging in an accelerated fashion compared with wild-type animals, resulting in heart failure and a reduced lifespan. In contrast, mice with low cardiac IGF1R signaling exhibited inferior cardiac function early in life, but superior cardiac performance during aging, and increased maximum lifespan, as well. Mechanistically, the late-life detrimental effects of IGF1R activation correlated with suppressed autophagic flux and impaired oxidative phosphorylation in the heart. Low IGF1R activity consistently improved myocardial bioenergetics and function of the aging heart in an autophagy-dependent manner. In humans, failing hearts, but not those with compensated hypertrophy, displayed exaggerated IGF1R expression and signaling activity. CONCLUSIONS: Our findings indicate that the relationship between IGF1R signaling and cardiac health is not linear, but rather biphasic. Hence, pharmacological inhibitors of the IGF1 pathway, albeit unsuitable for young individuals, might be worth considering in older adults.

Proteome characterisation of extracellular vesicles isolated from heart.

Cardiac intercellular communication is critical for heart function and often dysregulated in cardiovascular diseases. While cardiac extracellular vesicles (cEVs) are emerging mediators of signalling, their isolation remains a technical challenge hindering our understanding of cEV protein composition. Here, we utilised Langendorff-collagenase-based enzymatic perfusion and differential centrifugation to isolate cEVs from mouse heart (yield 3-6 µg/heart). cEVs are ∼200 nm, express classical EV markers (Cd63/81/9+ , Tsg101+ , Pdcd6ip/Alix+ ), and are depleted of blood (Alb/Fga/Hba) and cardiac damage markers (Mb, Tnnt2, Ldhb). Comparison with mechanically-derived EVs revealed greater detection of EV markers and decreased cardiac damage contaminants. Mass spectrometry-based proteomic profiling revealed 1721 proteins in cEVs, implicated in proteasomal and autophagic proteostasis, glycolysis, and fatty acid metabolism; essential functions often disrupted in cardiac pathologies. There was striking enrichment of 942 proteins in cEVs compared to mouse heart tissue - implicated in EV biogenesis, antioxidant activity, and lipid transport, suggesting active cargo selection and specialised function. Interestingly, cEVs contain marker proteins for cardiomyocytes, cardiac progenitors, B-cells, T-cells, macrophages, smooth muscle cells, endothelial cells, and cardiac fibroblasts, suggesting diverse cellular origin. We present a method of cEV isolation and provide insight into potential functions, enabling future studies into EV roles in cardiac physiology and disease.

FoxO1 is required for physiological cardiac hypertrophy induced by exercise but not by constitutively active PI3K.

The insulin-like growth factor 1 receptor (IGF1R) and phosphoinositide 3-kinase p110α (PI3K) are critical regulators of exercise-induced physiological cardiac hypertrophy and provide protection in experimental models of pathological remodeling and heart failure. Forkhead box class O1 (FoxO1) is a transcription factor that regulates cardiomyocyte hypertrophy downstream of IGF1R/PI3K activation in vitro, but its role in physiological hypertrophy in vivo was unknown. We generated cardiomyocyte-specific FoxO1 knockout (cKO) mice and assessed the phenotype under basal conditions and settings of physiological hypertrophy induced by 1) swim training or 2) cardiac-specific transgenic expression of constitutively active PI3K (caPI3KTg+). Under basal conditions, male and female cKO mice displayed mild interstitial fibrosis compared with control (CON) littermates, but no other signs of cardiac pathology were present. In response to exercise training, female CON mice displayed an increase (∼21%) in heart weight normalized to tibia length vs. untrained mice. Exercise-induced hypertrophy was blunted in cKO mice. Exercise increased cardiac Akt phosphorylation and IGF1R expression but was comparable between genotypes. However, differences in Foxo3a, Hsp70, and autophagy markers were identified in hearts of exercised cKO mice. Deletion of FoxO1 did not reduce cardiac hypertrophy in male or female caPI3KTg+ mice. Cardiac Akt and FoxO1 protein expressions were significantly reduced in hearts of caPI3KTg+ mice, which may represent a negative feedback mechanism from chronic caPI3K, and negate any further effect of reducing FoxO1 in the cKO. In summary, FoxO1 contributes to exercise-induced hypertrophy. This has important implications when one is considering FoxO1 as a target for treating the diseased heart.NEW & NOTEWORTHY Regulators of exercise-induced physiological cardiac hypertrophy and protection are considered promising targets for the treatment of heart failure. Unlike pathological hypertrophy, the transcriptional regulation of physiological hypertrophy has remained largely elusive. To our knowledge, this is the first study to show that the transcription factor FoxO1 is a critical mediator of exercise-induced cardiac hypertrophy. Given that exercise-induced hypertrophy is protective, this finding has important implications when one is considering FoxO1 as a target for treating the diseased heart.

Exercise training reveals micro-RNAs associated with improved cardiac function and electrophysiology in rats with heart failure after myocardial infarction.

AIMS: Endurance training improves aerobic fitness and cardiac function in individuals with heart failure. However, the underlying mechanisms are not well characterized. Exercise training could therefore act as a tool to discover novel targets for heart failure treatment. We aimed to associate changes in Ca2+ handling and electrophysiology with micro-RNA (miRNA) profile in exercise trained heart failure rats to establish which miRNAs induce heart failure-like effects in Ca2+ handling and electrophysiology. METHODS AND RESULTS: Post-myocardial infarction (MI) heart failure was induced in Sprague Dawley rats. Rats with MI were randomized to sedentary control (sed), moderate (mod)- or high-intensity (high) endurance training for 8 weeks. Exercise training improved cardiac function, Ca2+ handling and electrophysiology including reduced susceptibility to arrhythmia in an exercise intensity-dependent manner where high intensity gave a larger effect. Fifty-five miRNAs were significantly regulated (up or down) in MI-sed, of which 18 and 3 were changed towards Sham-sed in MI-high and MI-mod, respectively. Thereafter we experimentally altered expression of these "exercise-miRNAs" individually in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM) in the same direction as they were changed in MI. Of the "exercise-miRNAs", miR-214-3p prolonged AP duration, whereas miR-140 and miR-208a shortened AP duration. miR-497-5p prolonged Ca2+ release whereas miR-214-3p and miR-31a-5p prolonged Ca2+ decay. CONCLUSION: Using exercise training as a tool, we discovered that miR-214-3p, miR-497-5p, miR-31a-5p contribute to heart-failure like behaviour in Ca2+ handling and electrophysiology and could be potential treatment targets.

Gene therapy targeting cardiac phosphoinositide 3-kinase (p110α) attenuates cardiac remodeling in type 2 diabetes.

Diabetic cardiomyopathy is a distinct form of heart disease that represents a major cause of death and disability in diabetic patients, particularly, the more prevalent type 2 diabetes patient population. In the current study, we investigated whether administration of recombinant adeno-associated viral vectors carrying a constitutively active phosphoinositide 3-kinase (PI3K)(p110α) construct (rAAV6-caPI3K) at a clinically relevant time point attenuates diabetic cardiomyopathy in a preclinical type 2 diabetes (T2D) model. T2D was induced by a combination of a high-fat diet (42% energy intake from lipid) and low-dose streptozotocin (three consecutive intraperitoneal injections of 55 mg/kg body wt), and confirmed by increased body weight, mild hyperglycemia, and impaired glucose tolerance (all P < 0.05 vs. nondiabetic mice). After 18 wk of untreated diabetes, impaired left ventricular (LV) systolic dysfunction was evident, as confirmed by reduced fractional shortening and velocity of circumferential fiber shortening (Vcfc, all P < 0.01 vs. baseline measurement). A single tail vein injection of rAAV6-caPI3K gene therapy (2×1011vector genomes) was then administered. Mice were followed for an additional 8 wk before end point. A single injection of cardiac targeted rAAV6-caPI3K attenuated diabetes-induced cardiac remodeling by limiting cardiac fibrosis (reduced interstitial and perivascular collagen deposition, P < 0.01 vs. T2D mice) and cardiomyocyte hypertrophy (reduced cardiomyocyte size and Nppa gene expression, P < 0.001 and P < 0.05 vs. T2D mice, respectively). The diabetes-induced LV systolic dysfunction was reversed with rAAV6-caPI3K, as demonstrated by improved fractional shortening and velocity of circumferential fiber shortening (all P < 0.05 vs pre-AAV measurement). This cardioprotection occurred in combination with reduced LV reactive oxygen species (P < 0.05 vs. T2D mice) and an associated decrease in markers of endoplasmic reticulum stress (reduced Grp94 and Chop, all P < 0.05 vs. T2D mice). Together, our findings demonstrate that a cardiac-selective increase in PI3K(p110α), via rAAV6-caPI3K, attenuates T2D-induced diabetic cardiomyopathy, providing proof of concept for potential translation to the clinic.NEW & NOTEWORTHY Diabetes remains a major cause of death and disability worldwide (and its resultant heart failure burden), despite current care. The lack of existing management of heart failure in the context of the poorer prognosis of concomitant diabetes represents an unmet clinical need. In the present study, we now demonstrate that delayed intervention with PI3K gene therapy (rAAV6-caPI3K), administered as a single dose in mice with preexisting type 2 diabetes, attenuates several characteristics of diabetic cardiomyopathy, including diabetes-induced impairments in cardiac remodeling, oxidative stress, and function. Our discovery here contributes to the previous body of work, suggesting the cardioprotective effects of PI3K(p110α) could be a novel therapeutic approach to reduce the progression to heart failure and death in diabetes-affected patients.

Clusterin is regulated by IGF1-PI3K signaling in the heart: implications for biomarker and drug target discovery, and cardiotoxicity.

Open-access gene expression data sets provide a useful resource for identifying novel drug targets and biomarkers. The IGF1-PI3K pathway is a critical mediator of physiological cardiac enlargement/hypertrophy and protection. This study arose after mining a gene microarray data set from a previous study that compared heart tissue from cardiac-specific PI3K transgenic mouse models. The top-ranked candidate identified from the microarray data was clusterin. Clusterin has been proposed as a biomarker for multiple diseases including heart failure, and as a cancer drug target. Here, we show that clusterin gene expression is increased in hearts of transgenic mice with increased PI3K and decreased in mice with depressed cardiac PI3K. In vitro, clusterin secretion was elevated in media from neonatal rat ventricular myocytes treated with IGF1. Furthermore, by mining gene expression data from hearts during normal mouse postnatal growth, we also report an increase in clusterin expression with postnatal heart growth. Given we show that clusterin is regulated by the IGF1-PI3K pathway in the heart, and this pathway mediates physiological cardiac hypertrophy and cardioprotection, caution is required when considering clusterin as a biomarker for heart failure and as a cancer target. Mining pre-existing cardiac profiling data sets may be a useful approach to assess whether regulating new drug targets is likely to lead to cardiac damage/toxicity.

CORP: Practical tools for improving experimental design and reporting of laboratory studies of cardiovascular physiology and metabolism.

The exercise consisted of: 1) a short survey to acquire baseline data on current practices regarding the conduct of animal studies, 2) a series of presentations for promoting awareness and providing advice and practical tools for improving experimental design, and 3) a follow-up survey 12 mo later to assess whether practices had changed. The surveys were compulsory for responsible investigators (n = 16; paired data presented). Other investigators named on animal ethics applications were encouraged to participate (2017, total of 36 investigators; 2018, 37 investigators). The major findings to come from the exercise included 1) a willingness of investigators to make changes when provided with knowledge/tools and solutions that were relatively simple to implement (e.g., proportion of responsible investigators showing improved practices using a structured method for randomization was 0.44, 95% CI (0.19; 0.70), P = 0.003, and deidentifying drugs/interventions was 0.40, 95% CI (0.12; 0.68), P = 0.010); 2) resistance to change if this involved more personnel and time (e.g., as required for allocation concealment); and 3) evidence that changes to long-term practices ("habits") require time and follow-up. Improved practices could be verified based on changes in reporting within publications or documented evidence provided during laboratory visits. In summary, this exercise resulted in changed attitudes, practices, and reporting, but continued follow-up, monitoring, and incentives are required. Efforts to improve experimental rigor will reduce bias and will lead to findings with the greatest translational potential.NEW & NOTEWORTHY The goal of this exercise was to encourage preclinical researchers to improve the quality of their cardiac and metabolic animal studies by 1) increasing awareness of concerns, which can arise from suboptimal experimental designs; 2) providing knowledge, tools, and templates to overcome bias; and 3) conducting two short surveys over 12 mo to monitor change. Improved practices were identified for the uptake of structured methods for randomization, and de-identifying interventions/drugs.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/experimental-design-survey-training-practical-tools/.

Galectin-3 deficiency ameliorates fibrosis and remodeling in dilated cardiomyopathy mice with enhanced Mst1 signaling.

Dilated cardiomyopathy (DCM) is a major cause of heart failure without effective therapy. Fibrogenesis plays a key role in the development of DCM, but little is known of the expression of the profibrotic factor galectin-3 (Gal-3) and its role in DCM pathophysiology. In a mouse DCM model with transgenic (TG) overexpression of mammalian sterile 20-like kinase 1 (Mst1), we studied Gal-3 expression and effects of the Gal-3 inhibitor modified citrus pectin (MCP) or Gal-3 gene knockout (KO). Gal-3 deletion in TG mice (TG/KO) was achieved by crossbreeding Mst1-TG mice with Gal-3 KO mice. The DCM phenotype was assessed by echocardiography and micromanometry. Cardiac expression of Gal-3 and fibrosis were determined. The cardiac transcriptome was profiled by RNA sequencing. Mst1-TG mice at 3-8 mo of age exhibited upregulated expression of Gal-3 by ~40-fold. TG mice had dilatation of cardiac chambers, suppressed left ventricular (LV) ejection fraction, poor LV contractility and relaxation, a threefold increase in LV collagen content, and upregulated fibrotic genes. Four-month treatment with MCP showed no beneficial effects. Gal-3 deletion in Mst1-TG mice attenuated chamber dilatation, organ congestion, and fibrogenesis. RNA sequencing identified profound disturbances by Mst1 overexpression in the cardiac transcriptome, which largely remained in TG/KO hearts. Gal-3 deletion in Mst1-TG mice, however, partially reversed the dysregulated transcriptional signaling involving extracellular matrix remodeling and collagen formation. We conclude that cardiac Mst1 activation leads to marked Gal-3 upregulation and transcriptome disturbances in the heart. Gal-3 deficiency attenuated cardiac remodeling and fibrotic signaling. NEW & NOTEWORTHY We found in a transgenic mouse dilated cardiomyopathy (DCM) model a pronounced upregulation of galectin-3 in cardiomyocytes. Galectin-3 gene deletion reduced cardiac fibrosis and fibrotic gene profiles and ameliorated cardiac remodeling and dysfunction. These benefits of galectin-3 deletion were in contrast to the lack of effect of treatment with the galectin-3 inhibitor modified citrus pectin. Our study suggests that suppression of galectin-3 mRNA expression could be used to treat DCM with high cardiac galectin-3 content.

Multiple receptors converge on H2-Q10 to regulate NK and γδT-cell development.

Class Ib major histocompatibility complex (MHC) is an extended family of molecules, which demonstrate tissue-specific expression and presentation of monomorphic antigens. These characteristics tend to imbue class Ib MHC with unique functions. H2-Q10 is potentially one such molecule that is overexpressed in the liver but its immunological function is not known. We have previously shown that H2-Q10 is a ligand for the natural killer cell receptor Ly49C and now, using H2-Q10-deficient mice, we demonstrate that H2-Q10 can also stabilize the expression of Qa-1b. In the absence of H2-Q10, the development and maturation of conventional hepatic natural killer cells is disrupted. We also provide evidence that H2-Q10 is a new high affinity ligand for CD8αα and controls the development of liver-resident CD8αα γδT cells. These data demonstrate that H2-Q10 has multiple roles in the development of immune subsets and identify an overlap of recognition within the class Ib MHC that is likely to be relevant to the regulation of immunity.

Improving the quality of preclinical research echocardiography: observations, training, and guidelines for measurement.

Informal training in preclinical research may be a contributor to the poor reproducibility of preclinical cardiology research and low rates of translation into clinical research and practice. Mouse echocardiography is a widely used technique to assess cardiac structure and function in drug intervention studies using disease models. The interobserver variability of clinical echocardiographic measurements has been shown to improve with formalized training, but preclinical echocardiography lacks similarly critical standardization of training. The aims of this investigation were to assess the interobserver variability of echocardiographic measurements from studies in mice and address any technical impediments to reproducibility by implementing standardized guidelines through formalized training. In this prospective, single-site, observational cohort study, 13 scientists performing preclinical echocardiographic image analysis were assessed for measurement of short-axis M-mode-derived dimensions and calculated left ventricular (LV) mass. Ten M-mode images of mouse hearts acquired and analyzed by an expert researcher with a spectrum of LV mass were selected for assessment and validated by autopsy weight. After the initial observation, a structured formal training program was introduced, and accuracy and reproducibility were reevaluated. Mean absolute percentage error for expert-calculated LV mass was 6 ± 4% compared with autopsy LV mass and 25 ± 21% for participants before training. Standardized formal training improved participant mean absolute percentage error by ~30% relative to expert-calculated LV mass ( P < 0.001). Participants initially categorized with high-range error (25-45%) improved to low-moderate error ranges (<15-25%). This report reveals an example of technical skill training insufficiency likely endemic to preclinical research and provides validated guidelines for echocardiographic measurement for adaptation to formalized in-training programs. NEW & NOTEWORTHY The informal training common to academic/research institutions may be a contributor to the relatively poor reproducibility observed for preclinical cardiac research. In our observation of echocardiography analysis in murine models, we present evidence of moderate interobserver variability in standard preclinical research practice at an Australian heart research institute. These observations give rise to our recommendations for practical guidelines for echocardiography analysis in an adaptable approach to general preclinical research skill training. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/preclinical-echocardiography-training-and-guidelines/ .

Upregulated galectin-3 is not a critical disease mediator of cardiomyopathy induced by ß2-adrenoceptor overexpression.

Preclinical studies have demonstrated that anti-galectin-3 (Gal-3) interventions are effective in attenuating cardiac remodeling, fibrosis, and dysfunction. We determined, in a transgenic (TG) mouse model of fibrotic cardiomyopathy, whether Gal-3 expression was elevated and whether Gal-3 played a critical role in disease development. We studied mice with fibrotic cardiomyopathy attributable to cardiac overexpression of human ß2-adrenoceptors (ß2-TG). Cardiac expression levels of Gal-3 and fibrotic or inflammatory genes were determined. The effect of Gal-3 inhibition in ß2-TG mice was studied by treatment with Gal-3 inhibitors ( N-acetyllactosamine and modified citrus pectin) or by deletion of Gal-3 through crossing ß2-TG and Gal-3 knockout mice. Changes in cardiomyopathy phenotypes were assessed by echocardiography and biochemical assays. In ß2-TG mice at 3, 6, and 9 mo of age, upregulation of Gal-3 expression was observed at mRNA (~6- to 15-fold) and protein (~4- to 8-fold) levels. Treatment of ß2-TG mice with N-acetyllactosamine (3 wk) or modified citrus pectin (3 mo) did not reverse cardiac fibrosis, inflammation, and cardiomyopathy. Similarly, Gal-3 gene deletion in ß2-TG mice aged 3 and 9 mo did not rescue the cardiomyopathy phenotype. In conclusion, the ß2-TG model of cardiomyopathy showed a robust upregulation of Gal-3 that correlated with disease severity, but Gal-3 inhibitors or Gal-3 gene deletion had no effect in halting myocardial fibrosis, remodeling, and dysfunction. Gal-3 may not be critical for cardiac fibrogenesis and remodeling in this cardiomyopathy model. NEW & NOTEWORTHY We showed a robust upregulation of cardiac galectin-3 (Gal-3) expression in a mouse model of cardiomyopathy attributable to cardiomyocyte-restricted transgenic activation of ß2-adrenoceptors. However, pharmacological and genetic inhibition of Gal-3 did not confer benefit in this model, implying that Gal-3 may not be a critical disease mediator of cardiac remodeling in this model.

Distinct lipidomic profiles in models of physiological and pathological cardiac remodeling, and potential therapeutic strategies.

Cardiac myocyte membranes contain lipids which remodel dramatically in response to heart growth and remodeling. Lipid species have both structural and functional roles. Physiological and pathological cardiac remodeling have very distinct phenotypes, and the identification of molecular differences represent avenues for therapeutic interventions. Whether the abundance of specific lipid classes is different in physiological and pathological models was largely unknown. The aim of this study was to determine whether distinct lipids are regulated in settings of physiological and pathological remodeling, and if so, whether modulation of differentially regulated lipids could modulate heart size and function. Lipidomic profiling was performed on cardiac-specific transgenic mice with 1) physiological cardiac hypertrophy due to increased Insulin-like Growth Factor 1 (IGF1) receptor or Phosphoinositide 3-Kinase (PI3K) signaling, 2) small hearts due to depressed PI3K signaling (dnPI3K), and 3) failing hearts due to dilated cardiomyopathy (DCM). In hearts of dnPI3K and DCM mice, several phospholipids (plasmalogens) were decreased and sphingolipids increased compared to mice with physiological hypertrophy. To assess whether restoration of plasmalogens could restore heart size or cardiac function, dnPI3K and DCM mice were administered batyl alcohol (BA; precursor to plasmalogen biosynthesis) in the diet for 16weeks. BA supplementation increased a major plasmalogen species (p18:0) in the heart but had no effect on heart size or function. This may be due to the concurrent reduction in other plasmalogen species (p16:0 and p18:1) with BA. Here we show that lipid species are differentially regulated in settings of physiological and pathological remodeling. Restoration of lipid species in the failing heart warrants further examination.

Gene delivery of medium chain acyl-coenzyme A dehydrogenase induces physiological cardiac hypertrophy and protects against pathological remodelling.

We previously showed that medium chain acyl-coenzyme A dehydrogenase (MCAD, key regulator of fatty acid oxidation) is positively modulated in the heart by the cardioprotective kinase, phosphoinositide 3-kinase (PI3K(p110α)). Disturbances in cardiac metabolism are a feature of heart failure (HF) patients and targeting metabolic defects is considered a potential therapeutic approach. The specific role of MCAD in the adult heart is unknown. To examine the role of MCAD in the heart and to assess the therapeutic potential of increasing MCAD in the failing heart, we developed a gene therapy tool using recombinant adeno-associated viral vectors (rAAV) encoding MCAD. We hypothesised that increasing MCAD expression may recapitulate the cardioprotective properties of PI3K(p110α). rAAV6:MCAD or rAAV6:control was delivered to healthy adult mice and to mice with pre-existing pathological hypertrophy and cardiac dysfunction due to transverse aortic constriction (TAC). In healthy mice, rAAV6:MCAD induced physiological hypertrophy (increase in heart size, normal systolic function and increased capillary density). In response to TAC (~15 weeks), heart weight/tibia length increased by ~60% in control mice and ~45% in rAAV6:MCAD mice compared with sham. This was associated with an increase in cardiomyocyte cross-sectional area in both TAC groups which was similar. However, hypertrophy in TAC rAAV6:MCAD mice was associated with less fibrosis, a trend for increased capillary density and a more favourable molecular profile compared with TAC rAAV6:control mice. In summary, MCAD induced physiological cardiac hypertrophy in healthy adult mice and attenuated features of pathological remodelling in a cardiac disease model.

Adeno-Associated Virus Gene Therapy: Translational Progress and Future Prospects in the Treatment of Heart Failure.

Despite advances in treatment over the past decade, heart failure remains a significant public health burden and a leading cause of death in the developed world. Gene therapy provides a promising approach for preventing and reversing cardiac abnormalities, however, clinical application has shown limited success to date. A substantial effort is being invested into the development of recombinant adeno-associated viruses (AAVs) for cardiac gene therapy as AAV gene therapy offers a high safety profile and provides sustained and efficient transgene expression following a once-off administration. Due to the physiological, anatomical and genetic similarities between large animals and humans, preclinical studies using large animal models for AAV gene therapy are crucial stepping stones between the laboratory and the clinic. Many molecular targets selected to treat heart failure using AAV gene therapy have been chosen because of their potential to regulate and restore cardiac contractility. Other genes targeted with AAV are involved with regulating angiogenesis, beta-adrenergic sensitivity, inflammation, physiological signalling and metabolism. While significant progress continues to be made in the field of AAV cardiac gene therapy, challenges remain in overcoming host neutralising antibodies, improving AAV vector cardiac-transduction efficiency and selectivity, and optimising the dose, route and method of delivery.

Phosphoinositide 3-kinase (p110α) gene delivery limits diabetes-induced cardiac NADPH oxidase and cardiomyopathy in a mouse model with established diastolic dysfunction.

Phosphoinositide 3-kinase [PI3K (p110α)] is able to negatively regulate the diabetes-induced increase in NADPH oxidase in the heart. Patients affected by diabetes exhibit significant cardiovascular morbidity and mortality, at least in part due to a cardiomyopathy characterized by oxidative stress and left ventricular (LV) dysfunction. Thus, PI3K (p110α) may represent a novel approach to protect the heart from diabetes-induced cardiac oxidative stress and dysfunction. In the present study, we investigated the therapeutic potential of a delayed intervention with cardiac-targeted PI3K gene therapy, administered to mice with established diabetes-induced LV diastolic dysfunction. Diabetes was induced in 6-week-old male mice by streptozotocin (STZ). After 8 weeks of untreated diabetes, LV diastolic dysfunction was confirmed by a reduction in echocardiography-derived transmitral E/A ratio. Diabetic and non-diabetic mice were randomly allocated to receive either recombinant adeno-associated viral vector-6 carrying a constitutively-active PI3K construct (recombinant adeno-associated-virus 6-constitutively active PI3K (p110α) (caPI3K) (rAAV6-caPI3K), single i.v. injection, 2 × 1011 vector genomes) or null vector, and were followed for a further 6 or 8 weeks. At study endpoint, diabetes-induced LV dysfunction was significantly attenuated by a single administration of rAAV6-caPI3K, administered 8 weeks after the induction of diabetes. Diabetes-induced impairments in each of LV NADPH oxidase, endoplasmic reticulum (ER) stress, apoptosis, cardiac fibrosis and cardiomyocyte hypertrophy, in addition to LV systolic dysfunction, were attenuated by delayed intervention with rAAV6-caPI3K. Hence, our demonstration that cardiac-targeted PI3K (p110α) gene therapy limits diabetes-induced up-regulation of NADPH oxidase and cardiac remodelling suggests new insights into promising approaches for the treatment of diabetic cardiomyopathy, at a clinically relevant time point (after diastolic dysfunction is manifested).

Identification of miR-34 regulatory networks in settings of disease and antimiR-therapy: Implications for treating cardiac pathology and other diseases.

Expression of the miR-34 family (miR-34a, -34b, -34c) is elevated in settings of heart disease, and inhibition with antimiR-34a/antimiR-34 has emerged as a promising therapeutic strategy. Under chronic cardiac disease settings, targeting the entire miR-34 family is more effective than targeting miR-34a alone. The identification of transcription factor (TF)-miRNA regulatory networks has added complexity to understanding the therapeutic potential of miRNA-based therapies. Here, we sought to determine whether antimiR-34 targets secondary miRNAs via TFs which could contribute to antimiR-34-mediated protection. Using miRNA-Seq we identified differentially regulated miRNAs in hearts from mice with cardiac pathology due to transverse aortic constriction (TAC), and focused on miRNAs which were also regulated by antimiR-34. Two clusters of stress-responsive miRNAs were classified as "pathological" and "cardioprotective," respectively. Using ChIPBase we identified 45 TF binding sites on the promoters of "pathological" and "cardioprotective" miRNAs, and 5 represented direct targets of miR-34, with the capacity to regulate other miRNAs. Knockdown studies in a cardiomyoblast cell line demonstrated that expression of 2 "pathological" miRNAs (let-7e, miR-31) was regulated by one of the identified TFs. Furthermore, by qPCR we confirmed that expression of let-7e and miR-31 was lower in hearts from antimiR-34 treated TAC mice; this may explain why targeting the entire miR-34 family is more effective than targeting miR-34a alone. Finally, we showed that Acsl4 (a common target of miR-34, let-7e and miR-31) was increased in hearts from TAC antimiR-34 treated mice. In summary, antimiR-34 regulates the expression of other miRNAs and this has implications for drug development.

The IGF1-PI3K-Akt Signaling Pathway in Mediating Exercise-Induced Cardiac Hypertrophy and Protection.

Regular physical activity or exercise training can lead to heart enlargement known as cardiac hypertrophy. Cardiac hypertrophy is broadly defined as an increase in heart mass. In adults, cardiac hypertrophy is often considered a poor prognostic sign because it often progresses to heart failure. Heart enlargement in a setting of cardiac disease is referred to as pathological cardiac hypertrophy and is typically characterized by cell death and depressed cardiac function. By contrast, physiological cardiac hypertrophy, as occurs in response to chronic exercise training (i.e. the 'athlete's heart'), is associated with normal or enhanced cardiac function. The following chapter describes the morphologically distinct types of heart growth, and the key role of the insulin-like growth factor 1 (IGF1) - phosphoinositide 3-kinase (PI3K)-Akt signaling pathway in regulating exercise-induced physiological cardiac hypertrophy and cardiac protection. Finally we summarize therapeutic approaches that target the IGF1-PI3K-Akt signaling pathway which are showing promise in preclinical models of heart disease.

Expressing an inhibitor of PLCß1b sustains contractile function following pressure overload.

The activity of phospholipase Cß1b (PLCß1b) is selectively elevated in failing myocardium and cardiac expression of PLCß1b causes contractile dysfunction. PLCß1b can be selectively inhibited by expressing a peptide inhibitor that prevents sarcolemmal localization. The inhibitory peptide, PLCß1b-CT was expressed in heart from a mini-gene using adeno-associated virus (rAAV6-PLCß1b-CT). rAAV6-PLCß1b-CT, or blank virus, was delivered IV (4×10(9)vg/g body weight) and trans-aortic-constriction (TAC) or sham-operation was performed 8weeks later. Expression of PLCß1b-CT prevented the loss of contractile function, eliminated lung congestion and improved survival following TAC with either a 'moderate' or 'severe' pressure gradient. Hypertrophy was attenuated but not eliminated. Expression of the PLCß1b-CT peptide 2-3weeks after TAC reduced contractile dysfunction and lung congestion, without limiting hypertrophy. PLCß1b inhibition ameliorates pathological responses following acute pressure overload. The targeting of PLCß1b to the sarcolemma provides the basis for the development of a new class of inotropic agent.