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.

A high-sucrose diet exacerbates the left ventricular phenotype in a high fat-fed streptozotocin rat model of diabetic cardiomyopathy.

Left ventricular (LV) dysfunction is an early, clinically detectable sign of cardiomyopathy in type 2 diabetes mellitus (T2DM) that precedes the development of symptomatic heart failure. Preclinical models of diabetic cardiomyopathy are essential to develop therapies that may prevent or delay the progression of heart failure. This study examined the molecular, structural, and functional cardiac phenotype of two rat models of T2DM induced by a high-fat diet (HFD) with a moderate- or high-sucrose content (containing 88.9 or 346 g/kg sucrose, respectively), plus administration of low-dose streptozotocin (STZ). At 8 wk of age, male Sprague-Dawley rats commenced a moderate- or high-sucrose HFD. Two weeks later, rats received low-dose STZ (35 mg/kg ip for 2 days) and remained on their respective diets. LV function was assessed by echocardiography 1 wk before end point. At 22 wk of age, blood and tissues were collected postmortem. Relative to chow-fed sham rats, diabetic rats on a moderate- or high-sucrose HFD displayed cardiac reactive oxygen species dysregulation, perivascular fibrosis, and impaired LV diastolic function. The diabetes-induced impact on LV adverse remodeling and diastolic dysfunction was more apparent when a high-sucrose HFD was superimposed on STZ. In conclusion, a high-sucrose HFD in combination with low-dose STZ produced a cardiac phenotype that more closely resembled T2DM-induced cardiomyopathy than STZ diabetic rats subjected to a moderate-sucrose HFD.NEW & NOTEWORTHY Left ventricular dysfunction and adverse remodeling were more pronounced in diabetic rats that received low-dose streptozotocin (STZ) and a high-sucrose high-fat diet (HFD) compared with those on a moderate-sucrose HFD in combination with STZ. Our findings highlight the importance of sucrose content in diet composition, particularly in preclinical studies of diabetic cardiomyopathy, and demonstrate that low-dose STZ combined with a high-sucrose HFD is an appropriate rodent model of cardiomyopathy in type 2 diabetes.

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.

Diastolic dysfunction in a pre-clinical model of diabetes is associated with changes in the cardiac non-myocyte cellular composition.

BACKGROUND: Diabetes is associated with a significantly elevated risk of cardiovascular disease and its specific pathophysiology remains unclear. Recent studies have changed our understanding of cardiac cellularity, with cellular changes accompanying diabetes yet to be examined in detail. This study aims to characterise the changes in the cardiac cellular landscape in murine diabetes to identify potential cellular protagonists in the diabetic heart. METHODS: Diabetes was induced in male FVB/N mice by low-dose streptozotocin and a high-fat diet for 26-weeks. Cardiac function was measured by echocardiography at endpoint. Flow cytometry was performed on cardiac ventricles as well as blood, spleen, and bone-marrow at endpoint from non-diabetic and diabetic mice. To validate flow cytometry results, immunofluorescence staining was conducted on left-ventricles of age-matched mice. RESULTS: Mice with diabetes exhibited hyperglycaemia and impaired glucose tolerance at endpoint. Echocardiography revealed reduced E:A and e':a' ratios in diabetic mice indicating diastolic dysfunction. Systolic function was not different between the experimental groups. Detailed examination of cardiac cellularity found resident mesenchymal cells (RMCs) were elevated as a result of diabetes, due to a marked increase in cardiac fibroblasts, while smooth muscle cells were reduced in proportion. Moreover, we found increased levels of Ly6Chi monocytes in both the heart and in the blood. Consistent with this, the proportion of bone-marrow haematopoietic stem cells were increased in diabetic mice. CONCLUSIONS: Murine diabetes results in distinct changes in cardiac cellularity. These changes-in particular increased levels of fibroblasts-offer a framework for understanding how cardiac cellularity changes in diabetes. The results also point to new cellular mechanisms in this context, which may further aid in development of pharmacotherapies to allay the progression of cardiomyopathy associated with diabetes.

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.

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.

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/ .

Estrogen receptor alpha deficiency in cardiomyocytes reprograms the heart-derived extracellular vesicle proteome and induces obesity in female mice.

Dysregulation of estrogen receptor alpha (ERα) has been linked with increased metabolic and cardiovascular disease risk. Here, we generate and characterize cardiomyocyte-specific ERα knockout (ERαHKO) mice to assess the role of ERα in the heart. The most striking phenotype was obesity in female ERαHKO but not male ERαHKO mice. Female ERαHKO mice showed cardiac dysfunction, mild glucose and insulin intolerance and reduced ERα gene expression in skeletal muscle and white adipose tissue. Transcriptomic, proteomic, lipidomic and metabolomic analyses revealed evidence of contractile and/or metabolic dysregulation in heart, skeletal muscle and white adipose tissue. We show that heart-derived extracellular vesicles from female ERαHKO mice contain a distinct proteome associated with lipid and metabolic regulation, and have the capacity to metabolically reprogram the target skeletal myocyte proteome with functional impacts on glycolytic capacity and reserve. This multi-omics study uncovers a cardiac-initiated and sex-specific cardiometabolic phenotype regulated by ERα and provides insights into extracellular vesicle-mediated interorgan communication.

Protein phosphatase 2A in the healthy and failing heart: New insights and therapeutic opportunities.

Protein phosphatases have emerged as critical regulators of phosphoprotein homeostasis in settings of health and disease. Protein phosphatase 2A (PP2A) encompasses a large subfamily of enzymes that remove phosphate groups from serine/threonine residues within phosphoproteins. The heterogeneity in PP2A structure, which arises from the grouping of different catalytic, scaffolding and regulatory subunit isoforms, creates distinct populations of catalytically active enzymes (i.e. holoenzymes) that localise to different parts of the cell. This structural complexity, combined with other regulatory mechanisms, such as interaction of PP2A heterotrimers with accessory proteins and post-translational modification of the catalytic and/or regulatory subunits, enables PP2A holoenzymes to target phosphoprotein substrates in a highly specific manner. In this review, we summarise the roles of PP2A in cardiac physiology and disease. PP2A modulates numerous processes that are vital for heart function including calcium handling, contractility, ß-adrenergic signalling, metabolism and transcription. Dysregulation of PP2A has been observed in human cardiac disease settings, including heart failure and atrial fibrillation. Efforts are underway, particularly in the cancer field, to develop therapeutics targeting PP2A activity. The development of small molecule activators of PP2A (SMAPs) and other compounds that selectively target specific PP2A holoenzymes (e.g. PP2A/B56α and PP2A/B56ε) will improve understanding of the function of different PP2A species in the heart, and may lead to the development of therapeutics for normalising aberrant protein phosphorylation in settings of cardiac remodelling and dysfunction.

A randomised preclinical trial of adrenaline use during cardiac arrest in mice.

Background: Adrenaline is routinely administered during cardiac arrest resuscitation. Using a novel murine model of cardiac arrest, this study evaluates the effects of adrenaline use on survival and end-organ injury. Methods: A total of 58 mice, including cardiac arrest (CA) and sham (SHAM) groups received intravenous potassium chloride either as a bolus (CA) or slow infusion (SHAM), inducing ECG-confirmed asystole (in CA only) for 4-minutes prior to intravenous adrenaline (+ADR;250 ul,32 ug/ml) or saline (-ADR;250 ul) and manual chest compressions (300 BPM) for 4-minutes. Mice with return of spontaneous circulation (ROSC) were assessed at 24- or 72-h timepoints. Results: Among animals that underwent CA, rates of ROSC (n = 21 (95 %) vs n = 14 (82 %), P = 0.18) and survival to the planned endpoint (n = 11 (50 %) vs n = 12 (71 %), P = 0.19) were similar when comparing those treated with (CA+ADR) and without (CA-ADR) adrenaline. However, in CA animals that initially achieved ROSC, subsequent mortality was approximately 3-fold greater with adrenaline treatment (48 % vs 14 %, P = 0.042). Among SHAM animals, adrenaline use had no impact on survival rates or other endpoints. Greater myocardial injury occurred in CA+ADR vs CA-ADR, with increased Hs-Troponin levels measured at 24- (26.0 ± 0.9 vs 9.4 ± 5.3 ng/mL, P = 0.015) and 72-h (20.9 ± 8.3 vs 5.0 ± 2.4 ng/mL, P = 0.012), associated with increased expression of pro-inflammatory and fibrotic genes within cardiac and renal tissue. Conclusion: Adrenaline did not improve ROSC or overall survival but following successful ROSC, its use resulted in 3-fold greater mortality rates. Adrenaline was also associated with increased myocardial injury, end-organ inflammation, and fibrosis. These findings underscore the need for further preclinical evaluation of alternate pharmacologic adjuncts for cardiopulmonary resuscitation that improve survival and limit end-organ injury.

Fine-tuning the cardiac O-GlcNAcylation regulatory enzymes governs the functional and structural phenotype of the diabetic heart.

AIMS: The glucose-driven enzymatic modification of myocardial proteins by the sugar moiety, ß-N-acetylglucosamine (O-GlcNAc), is increased in pre-clinical models of diabetes, implicating protein O-GlcNAc modification in diabetes-induced heart failure. Our aim was to specifically examine cardiac manipulation of the two regulatory enzymes of this process on the cardiac phenotype, in the presence and absence of diabetes, utilising cardiac-targeted recombinant-adeno-associated viral-vector-6 (rAAV6)-mediated gene delivery. METHODS AND RESULTS: In human myocardium, total protein O-GlcNAc modification was elevated in diabetic relative to non-diabetic patients, and correlated with left ventricular (LV) dysfunction. The impact of rAAV6-delivered O-GlcNAc transferase (rAAV6-OGT, facilitating protein O-GlcNAcylation), O-GlcNAcase (rAAV6-OGA, facilitating de-O-GlcNAcylation), and empty vector (null) were determined in non-diabetic and diabetic mice. In non-diabetic mice, rAAV6-OGT was sufficient to impair LV diastolic function and induce maladaptive cardiac remodelling, including cardiac fibrosis and increased Myh-7 and Nppa pro-hypertrophic gene expression, recapitulating characteristics of diabetic cardiomyopathy. In contrast, rAAV6-OGA (but not rAAV6-OGT) rescued LV diastolic function and adverse cardiac remodelling in diabetic mice. Molecular insights implicated impaired cardiac PI3K(p110α)-Akt signalling as a potential contributing mechanism to the detrimental consequences of rAAV6-OGT in vivo. In contrast, rAAV6-OGA preserved PI3K(p110α)-Akt signalling in diabetic mouse myocardium in vivo and prevented high glucose-induced impairments in mitochondrial respiration in human cardiomyocytes in vitro. CONCLUSION: Maladaptive protein O-GlcNAc modification is evident in human diabetic myocardium, and is a critical regulator of the diabetic heart phenotype. Selective targeting of cardiac protein O-GlcNAcylation to restore physiological O-GlcNAc balance may represent a novel therapeutic approach for diabetes-induced heart failure.

Old Drug, New Trick: Tilorone, a Broad-Spectrum Antiviral Drug as a Potential Anti-Fibrotic Therapeutic for the Diseased Heart.

Cardiac fibrosis is associated with most forms of cardiovascular disease. No reliable therapies targeting cardiac fibrosis are available, thus identifying novel drugs that can resolve or prevent fibrosis is needed. Tilorone, an antiviral agent, can prevent fibrosis in a mouse model of lung disease. We investigated the anti-fibrotic effects of tilorone in human cardiac fibroblasts in vitro by performing a radioisotopic assay for [3H]-proline incorporation as a proxy for collagen synthesis. Exploratory studies in human cardiac fibroblasts treated with tilorone (10 µM) showed a significant reduction in transforming growth factor-ß induced collagen synthesis compared to untreated fibroblasts. To determine if this finding could be recapitulated in vivo, mice with established pathological remodelling due to four weeks of transverse aortic constriction (TAC) were administered tilorone (50 mg/kg, i.p) or saline every third day for eight weeks. Treatment with tilorone was associated with attenuation of fibrosis (assessed by Masson's trichrome stain), a favourable cardiac gene expression profile and no further deterioration of cardiac systolic function determined by echocardiography compared to saline treated TAC mice. These data demonstrate that tilorone has anti-fibrotic actions in human cardiac fibroblasts and the adult mouse heart, and represents a potential novel therapy to treat fibrosis associated with heart failure.

Characterisation of the Myocardial Mitochondria Structural and Functional Phenotype in a Murine Model of Diabetic Cardiomyopathy.

People affected by diabetes are at an increased risk of developing heart failure than their non-diabetic counterparts, attributed in part to a distinct cardiac pathology termed diabetic cardiomyopathy. Mitochondrial dysfunction and excess reactive oxygen species (ROS) have been implicated in a range of diabetic complications and are a common feature of the diabetic heart. In this study, we sought to characterise impairments in mitochondrial structure and function in a recently described experimental mouse model of diabetic cardiomyopathy. Diabetes was induced in 6-week-old male FVB/N mice by the combination of three consecutive-daily injections of low-dose streptozotocin (STZ, each 55 mg/kg i.p.) and high-fat diet (42% fat from lipids) for 26 weeks. At study end, diabetic mice exhibited elevated blood glucose levels and impaired glucose tolerance, together with increases in both body weight gain and fat mass, replicating several aspects of human type 2 diabetes. The myocardial phenotype of diabetic mice included increased myocardial fibrosis and left ventricular (LV) diastolic dysfunction. Elevated LV superoxide levels were also evident. Diabetic mice exhibited a spectrum of LV mitochondrial changes, including decreased mitochondria area, increased levels of mitochondrial complex-III and complex-V protein abundance, and reduced complex-II oxygen consumption. In conclusion, these data suggest that the low-dose STZ-high fat experimental model replicates some of the mitochondrial changes seen in diabetes, and as such, this model may be useful to study treatments that target the mitochondria in diabetes.

Bone Morphogenetic Protein 7 Gene Delivery Improves Cardiac Structure and Function in a Murine Model of Diabetic Cardiomyopathy.

Diabetes is a major contributor to the increasing burden of heart failure prevalence globally, at least in part due to a disease process termed diabetic cardiomyopathy. Diabetic cardiomyopathy is characterised by cardiac structural changes that are caused by chronic exposure to the diabetic milieu. These structural changes are a major cause of left ventricular (LV) wall stiffness and the development of LV dysfunction. In the current study, we investigated the therapeutic potential of a cardiac-targeted bone morphogenetic protein 7 (BMP7) gene therapy, administered once diastolic dysfunction was present, mimicking the timeframe in which clinical management of the cardiomyopathy would likely be desired. Following 18 weeks of untreated diabetes, mice were administered with a single tail-vein injection of recombinant adeno-associated viral vector (AAV), containing the BMP7 gene, or null vector. Our data demonstrated, after 8 weeks of treatment, that rAAV6-BMP7 treatment exerted beneficial effects on LV functional and structural changes. Importantly, diabetes-induced LV dysfunction was significantly attenuated by a single administration of rAAV6-BMP7. This was associated with a reduction in cardiac fibrosis, cardiomyocyte hypertrophy and cardiomyocyte apoptosis. In conclusion, BMP7 gene therapy limited pathological remodelling in the diabetic heart, conferring an improvement in cardiac function. These findings provide insight for the potential development of treatment strategies urgently needed to delay or reverse LV pathological remodelling in the diabetic heart.

Corrigendum: Characterising an Alternative Murine Model of Diabetic Cardiomyopathy.

[This corrects the article DOI: 10.3389/fphys.2019.01395.].

Loss of the long non-coding RNA OIP5-AS1 exacerbates heart failure in a sex-specific manner.

Long non-coding RNAs (lncRNAs) have been demonstrated to influence numerous biological processes, being strongly implicated in the maintenance and physiological function of various tissues including the heart. The lncRNA OIP5-AS1 (1700020I14Rik/Cyrano) has been studied in several settings; however its role in cardiac pathologies remains mostly uncharacterized. Using a series of in vitro and ex vivo methods, we demonstrate that OIP5-AS1 is regulated during cardiac development in rodent and human models and in disease settings in mice. Using CRISPR, we engineered a global OIP5-AS1 knockout (KO) mouse and demonstrated that female KO mice develop exacerbated heart failure following cardiac pressure overload (transverse aortic constriction [TAC]) but male mice do not. RNA-sequencing of wild-type and KO hearts suggest that OIP5-AS1 regulates pathways that impact mitochondrial function. Thus, these findings highlight OIP5-AS1 as a gene of interest in sex-specific differences in mitochondrial function and development of heart failure.

Defining the Progression of Diabetic Cardiomyopathy in a Mouse Model of Type 1 Diabetes.

The incidence of diabetes and its association with increased cardiovascular disease risk represents a major health issue worldwide. Diabetes-induced hyperglycemia is implicated as a central driver of responses in the diabetic heart such as cardiomyocyte hypertrophy, fibrosis, and oxidative stress, termed diabetic cardiomyopathy. The onset of these responses in the setting of diabetes has not been studied to date. This study aimed to determine the time course of development of diabetic cardiomyopathy in a model of type 1 diabetes (T1D) in vivo. Diabetes was induced in 6-week-old male FVB/N mice via streptozotocin (55 mg/kg i.p. for 5 days; controls received citrate vehicle). At 2, 4, 8, 12, and 16 weeks of untreated diabetes, left ventricular (LV) function was assessed by echocardiography before post-mortem quantification of markers of LV cardiomyocyte hypertrophy, collagen deposition, DNA fragmentation, and changes in components of the hexosamine biosynthesis pathway (HBP) were assessed. Blood glucose and HbA1c levels were elevated by 2 weeks of diabetes. LV and muscle (gastrocnemius) weights were reduced from 8 weeks, whereas liver and kidney weights were increased from 2 and 4 weeks of diabetes, respectively. LV diastolic function declined with diabetes progression, demonstrated by a reduction in E/A ratio from 4 weeks of diabetes, and an increase in peak A-wave amplitude, deceleration time, and isovolumic relaxation time (IVRT) from 4-8 weeks of diabetes. Systemic and local inflammation (TNFα, IL-1ß, CD68) were increased with diabetes. The cardiomyocyte hypertrophic marker Nppa was increased from 8 weeks of diabetes while ß-myosin heavy chain was increased earlier, from 2 weeks of diabetes. LV fibrosis (picrosirius red; Ctgf and Tgf-ß gene expression) and DNA fragmentation (a marker of cardiomyocyte apoptosis) increased with diabetes progression. LV Nox2 and Cd36 expression were elevated after 16 weeks of diabetes. Markers of the LV HBP (Ogt, Oga, Gfat1/2 gene expression), and protein abundance of OGT and total O-GlcNAcylation, were increased by 16 weeks of diabetes. This is the first study to define the progression of cardiac markers contributing to the development of diabetic cardiomyopathy in a mouse model of T1D, confirming multiple pathways contribute to disease progression at various time points.

Characterising an Alternative Murine Model of Diabetic Cardiomyopathy.

The increasing burden of heart failure globally can be partly attributed to the increased prevalence of diabetes, and the subsequent development of a distinct form of heart failure known as diabetic cardiomyopathy. Despite this, effective treatment options have remained elusive, due partly to the lack of an experimental model that adequately mimics human disease. In the current study, we combined three consecutive daily injections of low-dose streptozotocin with high-fat diet, in order to recapitulate the long-term complications of diabetes, with a specific focus on the diabetic heart. At 26 weeks of diabetes, several metabolic changes were observed including elevated blood glucose, glycated haemoglobin, plasma insulin and plasma C-peptide. Further analysis of organs commonly affected by diabetes revealed diabetic nephropathy, underlined by renal functional and structural abnormalities, as well as progressive liver damage. In addition, this protocol led to robust left ventricular diastolic dysfunction at 26 weeks with preserved systolic function, a key characteristic of patients with type 2 diabetes-induced cardiomyopathy. These observations corresponded with cardiac structural changes, namely an increase in myocardial fibrosis, as well as activation of several cardiac signalling pathways previously implicated in disease progression. It is hoped that development of an appropriate model will help to understand some the pathophysiological mechanisms underlying the accelerated progression of diabetic complications, leading ultimately to more efficacious treatment options.