Expression Levels of the Tnni3k Gene in the Heart Are Highly Associated with Cardiac and Glucose Metabolism-Related Phenotypes and Functional Pathways.

BACKGROUND: Troponin-I interacting kinase encoded by the TNNI3K gene is expressed in nuclei and Z-discs of cardiomyocytes. Mutations in TNNI3K were identified in patients with cardiac conduction diseases, arrhythmias, and cardiomyopathy. METHODS: We performed cardiac gene expression, whole genome sequencing (WGS), and cardiac function analysis in 40 strains of BXD recombinant inbred mice derived from C57BL/6J (B6) and DBA/2J (D2) strains. Expression quantitative trait loci (eQTLs) mapping and gene enrichment analysis was performed, followed by validation of candidate Tnni3k-regulatory genes. RESULTS: WGS identified compound splicing and missense T659I Tnni3k variants in the D2 parent and some BXD strains (D allele) and these strains had significantly lower Tnni3k expression than those carrying wild-type Tnni3k (B allele). Expression levels of Tnni3k significantly correlated with multiple cardiac (heart rate, wall thickness, PR duration, and T amplitude) and metabolic (glucose levels and insulin resistance) phenotypes in BXDs. A significant cis-eQTL on chromosome 3 was identified for the regulation of Tnni3k expression. Furthermore, Tnni3k-correlated genes were primarily involved in cardiac and glucose metabolism-related functions and pathways. Genes Nodal, Gnas, Nfkb1, Bmpr2, Bmp7, Smad7, Acvr1b, Acvr2b, Chrd, Tgfb3, Irs1, and Ppp1cb were differentially expressed between the B and D alleles. CONCLUSIONS: Compound splicing and T659I Tnni3k variants reduce cardiac Tnni3k expression and Tnni3k levels are associated with cardiac and glucose metabolism-related phenotypes.

Direct reprogramming induces vascular regeneration post muscle ischemic injury.

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes-Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)-together with three reprogramming-helper genes-dominant-negative (DN)-TGFß, DN-Wnt8a, and acid ceramidase (AC)-to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.

Leveraging clinical epigenetics in heart failure with preserved ejection fraction: a call for individualized therapies.

Described as the 'single largest unmet need in cardiovascular medicine', heart failure with preserved ejection fraction (HFpEF) remains an untreatable disease currently representing 65% of new heart failure diagnoses. HFpEF is more frequent among women and associates with a poor prognosis and unsustainable healthcare costs. Moreover, the variability in HFpEF phenotypes amplifies complexity and difficulties in the approach. In this perspective, unveiling novel molecular targets is imperative. Epigenetic modifications-defined as changes of DNA, histones, and non-coding RNAs (ncRNAs)-represent a molecular framework through which the environment modulates gene expression. Epigenetic signals acquired over the lifetime lead to chromatin remodelling and affect transcriptional programmes underlying oxidative stress, inflammation, dysmetabolism, and maladaptive left ventricular remodelling, all conditions predisposing to HFpEF. The strong involvement of epigenetic signalling in this setting makes the epigenetic information relevant for diagnostic and therapeutic purposes in patients with HFpEF. The recent advances in high-throughput sequencing, computational epigenetics, and machine learning have enabled the identification of reliable epigenetic biomarkers in cardiovascular patients. Contrary to genetic tools, epigenetic biomarkers mirror the contribution of environmental cues and lifestyle changes and their reversible nature offers a promising opportunity to monitor disease states. The growing understanding of chromatin and ncRNAs biology has led to the development of several Food and Drug Administration approved 'epidrugs' (chromatin modifiers, mimics, anti-miRs) able to prevent transcriptional alterations underpinning left ventricular remodelling and HFpEF. In the present review, we discuss the importance of clinical epigenetics as a new tool to be employed for a personalized management of HFpEF.

Leveraging clinical epigenetics in heart failure with preserved ejection fraction: a call for individualized therapies.

Described as the 'single largest unmet need in cardiovascular medicine', heart failure with preserved ejection fraction (HFpEF) remains an untreatable disease currently representing 65% of new heart failure diagnoses. HFpEF is more frequent among women and associates with a poor prognosis and unsustainable healthcare costs. Moreover, the variability in HFpEF phenotypes amplifies complexity and difficulties in the approach. In this perspective, unveiling novel molecular targets is imperative. Epigenetic modifications-defined as changes of DNA, histones, and non-coding RNAs (ncRNAs)-represent a molecular framework through which the environment modulates gene expression. Epigenetic signals acquired over the lifetime lead to chromatin remodelling and affect transcriptional programmes underlying oxidative stress, inflammation, dysmetabolism, and maladaptive left ventricular remodelling, all conditions predisposing to HFpEF. The strong involvement of epigenetic signalling in this setting makes the epigenetic information relevant for diagnostic and therapeutic purposes in patients with HFpEF. The recent advances in high-throughput sequencing, computational epigenetics, and machine learning have enabled the identification of reliable epigenetic biomarkers in cardiovascular patients. Contrary to genetic tools, epigenetic biomarkers mirror the contribution of environmental cues and lifestyle changes and their reversible nature offers a promising opportunity to monitor disease states. The growing understanding of chromatin and ncRNAs biology has led to the development of several Food and Drug Administration approved 'epidrugs' (chromatin modifiers, mimics, anti-miRs) able to prevent transcriptional alterations underpinning left ventricular remodelling and HFpEF. In the present review, we discuss the importance of clinical epigenetics as a new tool to be employed for a personalized management of HFpEF.

Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix proteins and myofibroblast differentiation. Our previous findings have implicated resistin in cardiac fibrosis; however, the molecular mechanisms underlying this process are still unclear. Here we investigated the role of resistin in fibroblast-to-myofibroblast differentiation and elucidated the pathways involved in this process. Fibroblast-to-myofibroblast transdifferentiation was induced with resistin or TGFß1 in NIH-3T3 and adult cardiac fibroblasts. mRNA and protein expression of fibrotic markers were analyzed by qPCR and immunoblotting. Resistin-knockout mice, challenged with a high-fat diet (HFD) for 20 weeks to stimulate cardiac impairment, were analyzed for cardiac function and fibrosis using histologic and molecular methods. Cardiac fibroblasts stimulated with resistin displayed increased fibroblast-to-myofibroblast conversion, with increased levels of αSma, col1a1, Fn, Ccn2 and Mmp9, with remarkable differences in the actin network appearance. Mechanistically, resistin promotes fibroblast-to-myofibroblast transdifferentiation and fibrogenesis via JAK2/STAT3 and JNK/c-Jun signaling pathways, independent of TGFß1. Resistin-null mice challenged with HFD showed an improvement in cardiac function and a decrease in tissue fibrosis and reduced mRNA levels of fibrogenic markers. These findings are the first to delineate the role of resistin in the process of cardiac fibroblast-to-myofibroblast differentiation via JAK/STAT3 and JNK/c-Jun pathways, potentially leading to stimulation of cardiac fibrosis.

FTO-Dependent N6-Methyladenosine Regulates Cardiac Function During Remodeling and Repair.

BACKGROUND: Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. METHODS: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. RESULTS: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. CONCLUSIONS: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Deletion of delta-like 1 homologue accelerates fibroblast-myofibroblast differentiation and induces myocardial fibrosis.

AIMS: Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. METHODS AND RESULTS: Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-ß1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-ß1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-ß1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFß-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. CONCLUSION: These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFß/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFß signalling leads to chronic fibrosis.

HAX-1 regulates SERCA2a oxidation and degradation.

Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.

Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders.

Dysregulation of endoplasmic reticulum (ER) Ca(2+) homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) has emerged as a major contributor to ER stress. We pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction.

Empagliflozin Improves Left Ventricular Diastolic Dysfunction in a Genetic Model of Type 2 Diabetes.

PURPOSE: Cardiovascular (CV) diseases in type 2 diabetes (T2DM) represent an enormous burden with high mortality and morbidity. Sodium-glucose cotransporter 2 (SGLT2) inhibitors have recently emerged as a new antidiabetic class that improves glucose control, as well as body weight and blood pressure with no increased risk of hypoglycemia. The first CV outcome study terminated with empagliflozin, a specific SGLT2 inhibitor, has shown a reduction in CV mortality and in heart failure hospitalization, suggesting a beneficial impact on cardiac function which remains to be demonstrated. This study was designed to examine the chronic effect of empagliflozin on left ventricular (LV) systolic and diastolic functions in a genetic model of T2DM, ob/ob mice. METHODS AND RESULTS: Cardiac phenotype was characterized by echocardiography, in vivo hemodynamics, histology, and molecular profiling. Our results demonstrate that empagliflozin significantly lowered HbA1c and slightly reduced body weight compared to vehicle treatment with no obvious changes in insulin levels. Empagliflozin also improved LV maximum pressure and in vivo indices of diastolic function. While systolic function was grossly not affected in both groups at steady state, response to dobutamine stimulation was significantly improved in the empagliflozin-treated group, suggesting amelioration of contractile reserve. This was paralleled by an increase in phospholamban (PLN) phosphorylation and increased SERCA2a/PLN ratio, indicative of enhanced SERCA2a function, further supporting improved cardiac relaxation and diastolic function. In addition, empagliflozin reconciled diabetes-associated increase in MAPKs and dysregulated phosphorylation of IRS1 and Akt, leading to improvement in myocardial insulin sensitivity and glucose utilization. CONCLUSION: The data show that chronic treatment with empagliflozin improves diastolic function, preserves calcium handling and growth signaling pathways and attenuates myocardial insulin resistance in ob/ob mice, findings suggestive of a potential clinical utility for empagliflozin in the treatment of diastolic dysfunction.

Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy.

The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2 weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy.

Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair.

Adult mammalian hearts respond to injury with scar formation and not with cardiomyocyte proliferation, the cellular basis of regeneration. Although cardiogenic progenitor cells may maintain myocardial turnover, they do not give rise to a robust regenerative response. Here we show that extracellular periostin induced reentry of differentiated mammalian cardiomyocytes into the cell cycle. Periostin stimulated mononucleated cardiomyocytes to go through the full mitotic cell cycle. Periostin activated alphaV, beta1, beta3 and beta5 integrins located in the cardiomyocyte cell membrane. Activation of phosphatidylinositol-3-OH kinase was required for periostin-induced reentry of cardiomyocytes into the cell cycle and was sufficient for cell-cycle reentry in the absence of periostin. After myocardial infarction, periostin-induced cardiomyocyte cell-cycle reentry and mitosis were associated with improved ventricular remodeling and myocardial function, reduced fibrosis and infarct size, and increased angiogenesis. Thus, periostin and the pathway that it regulates may provide a target for innovative strategies to treat heart failure.

Boron Facilitates Amelioration of Hepatic Injury by the Osmolyte Glycine and Resolves Injury by Improving the Tissue Redox Homeostasis.

Background: Glycine is a conditional non-essential amino acid in human and other mammals. It is abundant in the liver and is known for a wide spectrum of characteristics including the antioxidant, antiinflammatory, immunomodulatory, and cryoprotective effects. The amino acid is a naturally occurring osmolyte compatible with protein surface interactions and has been reported in literature as a potent therapeutic immuno-nutrient for liver diseases such as alcoholic liver disease. Oral glycine administration protects ethanol-induced liver injury, improves serum and tissue lipid profile, and alleviates hepatic injury in various conditions. In recent years, sodium salt of boron (borax) has been reported for its beneficial effects on cellular stress, including the effects on cell survival, immunity, and tissue redox state. Incidentally both glycine and boron prevent apoptosis and promote cell survival under stress. Objective: This study investigates the beneficial effect of borax on liver protection by glycine. Methods: Briefly, liver toxicity was induced in rats by a single intraperitoneal injection of thioacetamide (400 mg/kg b. wt.). Results: Significant changes in oxidative stress and liver function test parameters, the molybdenum Fe-S flavin hydroxylase activity, nitric oxide and tissue histopathology were observed in thioacetamide treated positive control group. The changes were ameliorated both by glycine as well as borax, but the combinatorial treatment yielded a better response indicating the impact of boron supplementation on glycine mediated protection of liver injury in experimental animal model. Conclusions: The study has clinical implications as the hepatotoxicity caused by thioacetamide mimics features of hepatitis C infection in human.

Cardiac fibrogenesis: an immuno-metabolic perspective.

Cardiac fibrosis is a major and complex pathophysiological process that ultimately culminates in cardiac dysfunction and heart failure. This phenomenon includes not only the replacement of the damaged tissue by a fibrotic scar produced by activated fibroblasts/myofibroblasts but also a spatiotemporal alteration of the structural, biochemical, and biomechanical parameters in the ventricular wall, eliciting a reactive remodeling process. Though mechanical stress, post-infarct homeostatic imbalances, and neurohormonal activation are classically attributed to cardiac fibrosis, emerging evidence that supports the roles of immune system modulation, inflammation, and metabolic dysregulation in the initiation and progression of cardiac fibrogenesis has been reported. Adaptive changes, immune cell phenoconversions, and metabolic shifts in the cardiac nonmyocyte population provide initial protection, but persistent altered metabolic demand eventually contributes to adverse remodeling of the heart. Altered energy metabolism, mitochondrial dysfunction, various immune cells, immune mediators, and cross-talks between the immune cells and cardiomyocytes play crucial roles in orchestrating the transdifferentiation of fibroblasts and ensuing fibrotic remodeling of the heart. Manipulation of the metabolic plasticity, fibroblast-myofibroblast transition, and modulation of the immune response may hold promise for favorably modulating the fibrotic response following different cardiovascular pathological processes. Although the immunologic and metabolic perspectives of fibrosis in the heart are being reported in the literature, they lack a comprehensive sketch bridging these two arenas and illustrating the synchrony between them. This review aims to provide a comprehensive overview of the intricate relationship between different cardiac immune cells and metabolic pathways as well as summarizes the current understanding of the involvement of immune-metabolic pathways in cardiac fibrosis and attempts to identify some of the previously unaddressed questions that require further investigation. Moreover, the potential therapeutic strategies and emerging pharmacological interventions, including immune and metabolic modulators, that show promise in preventing or attenuating cardiac fibrosis and restoring cardiac function will be discussed.

Differential patterns of replacement and reactive fibrosis in pressure and volume overload are related to the propensity for ischaemia and involve resistin.

Pathological left ventricle (LV) hypertrophy (LVH) results in reactive and replacement fibrosis. Volume overload LVH (VOH) is less profibrotic than pressure overload LVH (POH). Studies attribute subendocardial fibrosis in POH to ischaemia, and reduced fibrosis in VOH to collagen degradation favouring dilatation. However, the mechanical origin of the relative lack of fibrosis in VOH is incompletely understood. We hypothesized that reduced ischaemia propensity in VOH compared to POH accounted for the reduced replacement fibrosis, along with reduced reactive fibrosis. Rats with POH (ascending aortic banding) evolved into either compensated-concentric POH (POH-CLVH) or dilated cardiomyopathy (POH-DCM); they were compared to VOH (aorta-caval fistula). We quantified LV fibrosis, structural and haemodynamic factors of ischaemia propensity, and the activation of profibrotic pathways. Fibrosis in POH-DCM was severe, subendocardial and subepicardial, in contrast with subendocardial fibrosis in POH-CLVH and nearly no fibrosis in VOH. The propensity for ischaemia was more important in POH versus VOH, explaining different patterns of replacement fibrosis. LV collagen synthesis and maturation, and matrix metalloproteinase-2 expression, were more important in POH. The angiotensin II-transforming growth-factor ß axis was enhanced in POH, and connective tissue growth factor (CTGF) was overexpressed in all types of LVH. LV resistin expression was markedly elevated in POH, mildly elevated in VOH and independently reflected chronic ischaemic injury after myocardial infarction. In vitro, resistin is induced by angiotensin II and induces CTGF in cardiomyocytes. Based on these findings, we conclude that a reduced ischaemia propensity and attenuated upstream reactive fibrotic pathways account for the attenuated fibrosis in VOH versus POH.

Bioactive Compounds and Cardiac Fibrosis: Current Insight and Future Prospect.

Cardiac fibrosis is a pathological condition characterized by excessive deposition of collagen and other extracellular matrix components in the heart. It is recognized as a major contributor to the development and progression of heart failure. Despite significant research efforts in characterizing and identifying key molecular mechanisms associated with myocardial fibrosis, effective treatment for this condition is still out of sight. In this regard, bioactive compounds have emerged as potential therapeutic antifibrotic agents due to their anti-inflammatory and antioxidant properties. These compounds exhibit the ability to modulate fibrogenic processes by inhibiting the production of extracellular matrix proteins involved in fibroblast to myofibroblast differentiation, or by promoting their breakdown. Extensive investigation of these bioactive compounds offers new possibilities for preventing or reducing cardiac fibrosis and its detrimental consequences. This comprehensive review aims to provide a thorough overview of the mechanisms underlying cardiac fibrosis, address the limitations of current treatment strategies, and specifically explore the potential of bioactive compounds as therapeutic interventions for the treatment and/or prevention of cardiac fibrosis.

Cardiac copper content and its relationship with heart physiology: Insights based on quantitative genetic and functional analyses using BXD family mice.

Background: Copper (Cu) is essential for the functioning of various enzymes involved in important cellular and physiological processes. Although critical for normal cardiac function, excessive accumulation, or deficiency of Cu in the myocardium is detrimental to the heart. Fluctuations in cardiac Cu content have been shown to cause cardiac pathologies and imbalance in systemic Cu metabolism. However, the genetic basis underlying cardiac Cu levels and their effects on heart traits remain to be understood. Representing the largest murine genetic reference population, BXD strains have been widely used to explore genotype-phenotype associations and identify quantitative trait loci (QTL) and candidate genes. Methods: Cardiac Cu concentration and heart function in BXD strains were measured, followed by QTL mapping. The candidate genes modulating Cu homeostasis in mice hearts were identified using a multi-criteria scoring/filtering approach. Results: Significant correlations were identified between cardiac Cu concentration and left ventricular (LV) internal diameter and volumes at end-diastole and end-systole, demonstrating that the BXDs with higher cardiac Cu levels have larger LV chamber. Conversely, cardiac Cu levels negatively correlated with LV posterior wall thickness, suggesting that lower Cu concentration in the heart is associated with LV hypertrophy. Genetic mapping identified six QTLs containing a total of 217 genes, which were further narrowed down to 21 genes that showed a significant association with cardiac Cu content in mice. Among those, Prex1 and Irx3 are the strongest candidates involved in cardiac Cu modulation. Conclusion: Cardiac Cu level is significantly correlated with heart chamber size and hypertrophy phenotypes in BXD mice, while being regulated by multiple genes in several QTLs. Prex1 and Irx3 may be involved in modulating Cu metabolism and its downstream effects and warrant further experimental and functional validations.

Resistin promotes cardiac hypertrophy via the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) and c-Jun N-terminal kinase/insulin receptor substrate 1 (JNK/IRS1) pathways.

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and ß-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-ß-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.

Na+/Ca2+ exchanger-1 protects against systolic failure in the Akitains2 model of diabetic cardiomyopathy via a CXCR4/NF-κB pathway.

Diabetic cardiomyopathy is characterized, in part, by calcium handling imbalances associated with ventricular dysfunction. The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) has been implicated as a compensatory mechanism in response to reduced contractility in the heart; however, its role in diabetic cardiomyopathy remains unknown. We aimed to fully characterize the Akita(ins2) murine model of type 1 diabetes through assessing cardiac function and NCX1 regulation. The CXCL12/CXCR4 chemokine axis is well described in its cardioprotective effects via progenitor cell recruitment postacute myocardial infarction; however, it also functions in regulating calcium dependent processes in the cardiac myocyte. We therefore investigated the potential impact of CXCR4 in diabetic cardiomyopathy. Cardiac performance in the Akita(ins2) mouse was monitored using echocardiography and in vivo pressure-volume analysis. The Akita(ins2) mouse is protected against ventricular systolic failure evident at both 5 and 12 mo of age. However, the preserved contractility was associated with a decreased sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a)/phospholamban ratio and increased NCX1 content. Direct myocardial injection of adenovirus encoding anti-sense NCX1 significantly decreased NCX1 expression and induced systolic failure in the Akita(ins2) mouse. CXCL12 and CXCR4 were both upregulated in the Akita(ins2) heart, along with an increase in IκB-α and NF-κB p65 phosphorylation. We demonstrated that CXCR4 activation upregulates NCX1 expression through a NF-κB-dependent signaling pathway in the cardiac myocyte. In conclusion, the Akita(ins2) type 1 diabetic model is protected against systolic failure due to increased NCX1 expression. In addition, our studies reveal a novel role of CXCR4 in the diabetic heart by regulating NCX1 expression via a NF-κB-dependent mechanism.