Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.     

IGF-1 deficiency resists cardiac hypertrophy and myocardial contractile dysfunction: role of microRNA-1 and microRNA-133a

This study was designed to examine the impact of insulin-like growth factor-1 (IGF-1) deficiency on abdominal aortic constriction (AAC)-induced cardiac geometric and functional changes with a focus on microRNA-1, 133a and 208, which are specially expressed in hearts and govern cardiac hypertrophy and stress-dependent cardiac growth. Liver-specific IGF-1-deficient (LID) and C57/BL6 mice were subject to AAC. Echocardiographic and cardiomyocyte function were assessed 4 wks later. Haematoxylin and eosin staining was used to monitor myocardial morphology. Western blot and real-time PCR were used to detect protein and miR expression, respectively. Neonatal rat cardiomyocytes (NRCMs) were transfected with miRs prior to IGF-1 exposure to initiate cell proliferation. Immunohistochemistry and [(3)H] Leucine incorporation were used to detect cell surface area and protein abundance. C57 mice subject to AAC displayed increased ventricular wall thickness, decreased left ventricular end diastolic and end systolic dimensions and elevated cardiomyocyte shortening capacity, all of which were attenuated in LID mice. In addition, IGF-1 deficiency mitigated AAC-induced increase in atrial natriuretic factor, GATA binding protein 4, glucose transporter 4 (GLUT4) and Akt phosphorylation. In contrast, neither AAC treatment nor IGF-1 deficiency affected glycogen synthase kinase 3b, mammalian target of rapamycin, the Glut-4 translocation mediator Akt substrate of 160 kD (AS160) and protein phosphatase. Levels of miR-1 and -133a (but not miR-208) were significantly attenuated by AAC in C57 but not LID mice. Transfection of miR-1 and -133a obliterated IGF-1-induced hypertrophic responses in NRCMs. Our data suggest that IGF-1 deficiency retards AAC-induced cardiac hypertrophic and contractile changes via alleviating down-regulation of miR-1 and miR-133a in response to left ventricular pressure overload.     

Cardiomyocyte overexpression of miR-27b induces cardiac hypertrophy and dysfunction in mice

Recent studies have begun to reveal critical roles of microRNAs (miRNAs) in the pathogenesis of cardiac hypertrophy and dysfunction. In this study, we tested whether a transforming growth factor-β (TGF-β)-regulated miRNA played a pivotal role in the development of cardiac hypertrophy and heart failure (HF). We observed that miR-27b was upregulated in hearts of cardiomyocyte-specific Smad4 knockout mice, which developed cardiac hypertrophy. In vitro experiments showed that the miR-27b expression could be inhibited by TGF-β1 and that its overexpression promoted hypertrophic cell growth, while the miR-27b suppression led to inhibition of the hypertrophic cell growth caused by phenylephrine (PE) treatment. Furthermore, the analysis of transgenic mice with cardiomyocyte-specific overexpression of miR-27b revealed that miR-27b overexpression was sufficient to induce cardiac hypertrophy and dysfunction. We validated the peroxisome proliferator-activated receptor-γ (PPAR-γ) as a direct target of miR-27b in cardiomyocyte. Consistently, the miR-27b transgenic mice displayed significantly lower levels of PPAR-γ than the control mice. Furthermore, in vivo silencing of miR-27b using a specific antagomir in a pressure-overload-induced mouse model of HF increased cardiac PPAR-γ expression, attenuated cardiac hypertrophy and dysfunction. The results of our study demonstrate that TGF-β1-regulated miR-27b is involved in the regulation of cardiac hypertrophy, and validate miR-27b as an efficient therapeutic target for cardiac diseases.     

Compensated hypertrophy of cardiac ventricles in aged transgenic FVB/N mice overexpressing calsequestrin

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed contractile parameters and hypertrophy in transgenic mice. To determine the long-term consequences of calsequestrin overexpression, the cardiac phenotype of young (2–3-months old) and aged (17 months old) transgenic FVB/N mice was characterized. Ventricular/body weight ratios, which were increased in young transgenics compared with wild-types, were unaltered with age. Left atria of aged transgenics exhibited enlargement and mineralization, but their ventricles did not display fibrosis, mineralization and other injuries. Although echocardiography suggested a time-dependent change in ventricular geometry and loading conditions in vivo, as well as an age-dependent reduction of left ventricular fractional shortening in transgenic mice, Langendorff-perfused hearts of young and aged transgenics indicated that there were no age-related reductions of contractile parameters (±dP/dt). Furthermore, neither genotype nor age altered lung/body weight ratios. Thus, our findings suggest that left ventricular performance in calsequestrin overexpressing mice becomes apparently depressed with age, but this depression is not associated with progressive reduction of left ventricular contractility and heart failure.     

High triiodothyronine levels induce myocardial hypertrophy via BAFF overexpression

Activated B cells contribute to heart diseases, and inhibition of B‐cell activating factor (BAFF) expression is an effective therapeutic target for heart diseases. Whether activated B cells participate in the development and progression of hyperthyroid heart disease, and what induces B cells activation in hyperthyroidism are unknown. The present study aimed to determine the roles of BAFF overexpression induced by high concentrations of triiodothyronine (T3) in the pathogenesis of hyperthyroid heart disease. Female C57BL/6J mice were subcutaneously injected with T3 for 6 weeks, and BAFF expression was inhibited using shRNA. Protein and mRNA expression of BAFF in mouse heart tissues evaluated via immunohistochemistry, western blotting and polymerase chain reaction (PCR). Proportions of B cells in mouse cardiac tissue lymphocytes were quantified via flow cytometry. Morphology and left ventricle function were assessed using pathological sections and echocardiography, respectively. Here, we demonstrate that compared with the control group, the proportion of myocardial B cells was larger in the T3 group; immunohistochemistry, western blotting and PCR analyses revealed increased protein and mRNA expression levels of TNF‐α and BAFF in heart tissues of the T3 group. Compared with the normal controls group, in the T3 group, the diameter of myocardial cells and some echocardiographic values significantly increased and hypertrophy and structural disorder were noticeable. Our results revealed that elevated levels of circulating T3 can promote the expression of BAFF in myocardial cells and can lead to B‐cell activation, an elevated inflammatory response and ventricular remodelling.     

Diabetes influences cardiac extracellular matrix remodelling after myocardial infarction and subsequent development of cardiac dysfunction

This study was conducted to examine the influence of acute streptozotocin‐induced diabetes on cardiac remodelling and function in mice subjected to myocardial infarction (MI) by coronary artery ligation. Echocardiography analysis indicated that diabetes induced deleterious cardiac functional changes as demonstrated by the negative differences of ejection fraction, fractional shortening, stroke volume, cardiac output and left ventricular volume 24 hrs after MI. Temporal analysis for up to 2 weeks after MI showed higher mortality in diabetic animals because of cardiac wall rupture. To examine extracellular matrix remodelling, we used fluorescent molecular tomography to conduct temporal studies and observed that total matrix metalloproteinase (MMP) activity in hearts was higher in diabetic animals at 7 and 14 days after MI, which correlated well with the degree of collagen deposition in the infarct area visualized by scanning electron microscopy. Gene arrays indicated temporal changes in expression of distinct MMP isoforms after 1 or 2 weeks after MI, particularly in diabetic mice. Temporal changes in cardiac performance were observed, with a trend of exaggerated dysfunction in diabetic mice up to 14 days after MI. Decreased radial and longitudinal systolic and diastolic strain rates were observed over 14 days after MI, and there was a trend towards altered strain rates in diabetic mouse hearts with dyssynchronous wall motion clearly evident. This correlated with increased collagen deposition in remote areas of these infarcted hearts indicated by Masson's trichrome staining. In summary, temporal changes in extracellular matrix remodelling correlated with exaggerated cardiac dysfunction in diabetic mice after MI.     

MiR-26a-5p alleviates cardiac hypertrophy and dysfunction via targeting ADAM17

Cardiac hypertrophy has been a high prevalence rate throughout the world. It has posed a big threat to public health due to limited therapeutic approaches. Previous studies showed that pathological cardiac hypertrophy was associated with autophagy, microRNAs (miRNA), and other signaling pathways, while the molecular mechanisms remain incompletely characterized. In this study, we used thoracic aortic constriction (TAC)-induced mice and angiotensin-II (Ang-II)-induced H9C2 cell line as cardiac hypertrophy model to investigate the role of miR-26a-5p in cardiac hypertrophy. We found that miR-26a-5p was downregulated in cardiac hypertrophy mice. Overexpression of miR-26a-5p by type 9 recombinant adeno-associated virus (rAAV9) reversed the heart hypertrophic manifestations. The phenotypes were also promoted by miR-26a-5p inhibitor in Ang-II-induced H9C2 cells. Through miRNA profile analysis and dual-luciferase reporter assay, ADAM17 was identified as a direct target of miR-26a-5p. Restored expression of ADAM17 disrupted the effect of miR-26a-5p on cardiac hypertrophy. To sum up, these results indicated that miR-26a-5p played an inhibitory role in cardiac hypertrophy and dysfunction via targeting ADAM17. The miR-26a-5p-ADAM17-cardiac hypertrophy axis provided special insight and a new molecular mechanism for a better understanding of cardiac hypertrophy disease, as well as the diagnostic and therapeutic practice.     

Time-dependent changes of the susceptibility of cardiac contractile function to hypoxia-reoxygenation after myocardial infarction in rats

In this study we analyzed the susceptibility of contractile function of the myocardium to hypoxia-reoxygenation after infarction. For this purpose, the contractility of isolated papillary muscles from rats was studied at high oxygen tension (pO₂ 80 kPa) and during hypoxia (pO₂ 3 kPa) with subsequent reoxygenation at variable intervals between 15 h and 9 weeks after permanent ligation of the left coronary artery. Hypoxic exposure reduced the contractile performance of the preparations to a similar extent in both groups. Notably, the contractility and, in particular, the relaxation rates recovered more completely from hypoxia in the hypertrophied myocardium of rats with coronary artery ligation than in sham-operated (SO) animals. The recovery of contractile function was improved maximally between 6 and 9 weeks after myocardial infarction (MI). The lower sensitivity of the (post)ischemic myocardium to hypoxia-reoxygenation correlated with enhanced left ventricular glutathione peroxidase (GSH-Px) activity (15 h to 9 weeks post-MI) and 2–3-fold increased expression levels (15 h to 6 weeks post-MI) of the 72 kDa heat shock protein (HSP72) in the papillary muscles. These findings suggest that the greater antioxidant potential and, possibly, stimulation of HSPs contribute to the sustained tolerance of the myocardium to hypoxia-reoxygenation injury after infarction.     

Impaired sarcoplasmic reticulum function leads to contractile dysfunction and cardiac hypertrophy

Sarcoplasmic reticulum (SR)-mediated Ca(2+) sequestration and release are important determinants of cardiac contractility. In end-stage heart failure SR dysfunction has been proposed to contribute to the impaired cardiac performance. In this study we tested the hypothesis that a targeted interference with SR function can be a primary cause of contractile impairment that in turn might alter cardiac gene expression and induce cardiac hypertrophy. To study this we developed a novel animal model in which ryanodine, a substance that alters SR Ca(2+) release, was added to the drinking water of mice. After 1 wk of treatment, in vivo hemodynamic measurements showed a 28% reduction in the maximum speed of contraction (+dP/dt(max)) and a 24% reduction in the maximum speed of relaxation (-dP/dt(max)). The slowing of cardiac relaxation was confirmed in isolated papillary muscles. The late phase of relaxation expressed as the time from 50% to 90% relaxation was prolonged by 22%. After 4 wk of ryanodine administration the animals had developed a significant cardiac hypertrophy that was most prominent in both atria (right atrium +115%, left atrium +100%, right ventricle +23%, and left ventricle +13%). This was accompanied by molecular changes including a threefold increase in atrial natriuretic factor mRNA and a sixfold increase in beta-myosin heavy chain mRNA. Sarcoplasmic endoplasmic reticulum Ca(2+) mRNA was reduced by 18%. These data suggest that selective impairment of SR function in vivo can induce changes in cardiac gene expression and promote cardiac growth.     

The protective effect of isosteviol sodium on cardiac function and myocardial remodelling in transverse aortic constriction rat

Pathological hypertrophy contributes to heart failure and there is not quite effective treatment to invert this process. Isosteviol has been shown to protect the heart against ischaemia-reperfusion injury and isoproterenol-induced cardiac hypertrophy, but its effect on pressure overload-induced cardiac hypertrophy is still unknown. Pressure overload induced by transverse aortic constriction (TAC) causes cardiac hypertrophy in rats to mimic the pathological condition in human. This study examined the effects of isosteviol sodium (STVNa) on cardiac hypertrophy by the TAC model and cellular assays in vitro. Cardiac function test, electrocardiogram analysis and histological analysis were conducted. The effects of STVNa on calcium transient of the adult rat ventricular cells and the proliferation of neonatal rat cardiac fibroblasts were also studied in vitro. Cardiac hypertrophy was observed after 3-week TAC while the extensive cardiac dysfunction and electronic remodelling were observed after 9-week TAC. Both STVNa and sildenafil (positive drug) treatment reversed the two process, but STVNa appeared to be more superior in some aspects and did not change calcium transient considerably. STVNa also reversed TAC-induced cardiac fibrosis in vivo and TGF-β1-induced fibroblast proliferation in vitro. Moreover, STVNa, but not sildenafil, reversed impairment of the autonomic nervous system induced by 9-week TAC.     

Acute lipoprotein lipase deletion in adult mice leads to dyslipidemia and cardiac dysfunction

The most energy-requiring organ in the body, the cardiac muscle, relies primarily on lipoprotein-derived fatty acids. Prenatal loss of cardiac lipoprotein lipase (LPL) leads to hypertriglyceridemia, but no cardiac dysfunction, in young mice. Cardiac specific loss of LPL in 8-wk-old mice was produced by a 2-wk tamoxifen treatment of MerCreMer (MCM)/Lpl(flox/flox) mice. LPL gene deletion was confirmed by PCR analysis, and LPL mRNA expression was reduced by approximately 70%. One week after tamoxifen was completed, triglyceride was increased with LPL deletion, 162 +/- 53 vs. 91 +/- 21 mg/dl, P < 0.01. Tamoxifen treatment of Lpl(flox/flox) mice did not cause a significant increase in triglyceride levels. Four weeks after tamoxifen, MCM/Lpl(flox/flox) mice had triglyceride levels of 190 +/- 27 mg/dl, similar to those of mice with prenatal LPL deletion. One week after the tamoxifen, MCM/Lpl(flox/flox), but not Lpl(flox/flox), mice had decreases in carnitine palmitoyl transferase I mRNA (18%) and pyruvate dehydrogenase kinase 4 mRNA (38%). These changes in gene expression became more robust with time. Acute loss of LPL decreased ejection fraction and increased mRNA levels for atrial natriuretic factor. Our studies show that acute loss of LPL can be produced and leads to rapid alteration in gene expression and cardiac dysfunction.     

C1q/TNF-related protein-9 promotes macrophage polarization and improves cardiac dysfunction after myocardial infarction

The timely regulation of inflammatory M1 macrophage polarization toward regenerative M2 macrophages suggests the possibility of immunotherapy after myocardial infarction (MI). C1q/TNF-related protein-9 (CTRP9) has anti-inflammatory effects and can ameliorate heart function in mice after long-term myocardial infarction. The role of CTRP9 in macrophage polarization remains completely unclear. This study determined whether CTRP9 can preserve post-MI early cardiac function through the regulation of macrophage polarization. In the present study, an adenovirus-delivered CTRP9 supplement promoted macrophage polarization at Day 3 post MI and improved cardiac function at Day 7 post MI. Pretreatment with gCTRP9 promoted the M1 to M2 polarization transition and attenuated inflammation after lipopolysaccharide + interferon-γ stimulation; the effects were partly abrogated by the adenosine monophosphate kinase (AMPK) inhibitor compound C and were obviously reinforced by pyrrolidine dithiocarbamate, a nuclear factor-κB (NF-κB) inhibitor. Meanwhile, CTPR9 markedly reduced the expression of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and NF-κB p65 phosphorylation by promoting AMPK phosphorylation in vivo and in vitro. Moreover, the competitive binding of gCTRP9 and LPS to the myeloid differentiation protein 2 (MD2)/TLR4 complex was associated with direct binding to MD2, thereby inhibiting the downstream signaling molecule MyD88. Taken together, we demonstrated that CTRP9 improved post-MI early cardiac function, at least in part, by modulating M1/M2 macrophage polarization, largely via the TLR4/MD2/MyD88 and AMPK-NF-κB pathways.     

Knockout of MYOM1 in human cardiomyocytes leads to myocardial atrophy via impairing calcium homeostasis

Myomesin-1 (encoded by MYOM1 gene) is expressed in almost all cross-striated muscles, whose family (together with myomesin-2 and myomesin-3) helps to cross-link adjacent myosin to form the M-line in myofibrils. However, little is known about its biological function, causal relationship and mechanisms underlying the MYOM1-related myopathies (especially in the heart). Regrettably, there is no MYMO1 knockout model for its study so far. A better and further understanding of MYOM1 biology is urgently needed. Here, we used CRISPR/Cas9 gene-editing technology to establish an MYOM1 knockout human embryonic stem cell line (MYOM1^−/− hESC), which was then differentiated into myomesin-1 deficient cardiomyocytes (MYOM1^−/− hESC-CMs) in vitro. We found that myomesin-1 plays an important role in sarcomere assembly, contractility regulation and cardiomyocytes development. Moreover, myomesin-1-deficient hESC-CMs can recapitulate myocardial atrophy phenotype in vitro. Based on this model, not only the biological function of MYOM1, but also the aetiology, pathogenesis, and potential treatments of myocardial atrophy caused by myomesin-1 deficiency can be studied.     

Transgenic overexpression of cardiac actin in the mouse heart suggests coregulation of cardiac,skeletal and vascular actin expression

Previous studies have shown that depletion of cardiac actin by targeted disruption is associated with increased expression of alternative actins in the mouse heart. Here we have studied the effects of transgenic overexpression of cardiac actin using the -myosin heavy chain promoter. Lines carrying 7 or 8 copies of the transgene showed a 2-fold increase in cardiac actin mRNA and also displayed decreased expression of skeletal and vascular actin in their hearts. In contrast, a line with more than 250 copies of the transgene did not show a similar decrease in the expression of skeletal and vascular actin despite a 3-fold increase in cardiac actin mRNA. While the low copy number transgenic mice displayed hearts that were similar to non-transgenic controls, the high copy number transgenic line showed larger hearts with distinct atrial enlargement and cardiomyocyte hypertrophy. Further, while the low copy number transgenic mouse hearts were mildly hypocontractile when compared with non-transgenic mouse hearts, the high copy number transgenic mouse hearts were significantly so. We conclude that in the presence of a small number of copies of the cardiac actin transgene, homeostatic mechanisms involved in maintaining actin levels are active and negatively regulate skeletal and vascular actin levels in the heart in response to increased expression of cardiac actin. However, these putative mechanisms are either inoperative in the high copy number transgenic line or are countered by the enhanced expression of skeletal and vascular actin during cardiomyocyte hypertrophy.     

Mitophagy‐regulated mitochondrial health strongly protects the heart against cardiac dysfunction after acute myocardial infarction

Autophagy including mitophagy serves as an important regulatory mechanism in the heart to maintain the cellular homeostasis and to protect against heart damages caused by myocardial infarction (MI). The current study aims to dissect roles of general autophagy and specific mitophagy in regulating cardiac function after MI. By using Beclin1^+/−, Fundc1 knockout (KO) and Fundc1 transgenic (TG) mouse models, combined with starvation and MI models, we found that Fundc1 KO caused more severe mitochondrial and cardiac dysfunction damages than Beclin1^+/− after MI. Interestingly, Beclin1^+/− caused notable decrease of total autophagy without detectable change to mitophagy, and Fundc1 KO markedly suppressed mitophagy but did not change the total autophagy activity. In contrast, starvation increased total autophagy without changing mitophagy while Fundc1 TG elevated total autophagy and mitophagy in mouse hearts. As a result, Fundc1 TG provided much stronger protective effects than starvation after MI. Moreover, Beclin1^+/−/Fundc1 TG showed increased total autophagy and mitophagy to a level comparable to Fundc1 TG per se, and completely reversed Beclin1^+/−‐caused aggravation of mitochondrial and cardiac injury after MI. Our results reveal that mitophagy but not general autophagy contributes predominantly to the cardiac protective effect through regulating mitochondrial function.     

IDH2 deficiency promotes mitochondrial dysfunction and cardiac hypertrophy in mice

Cardiac hypertrophy, a risk factor for heart failure, is associated with enhanced oxidative stress in the mitochondria, resulting from high levels of reactive oxygen species (ROS). The balance between ROS generation and ROS detoxification dictates ROS levels. As such, disruption of these processes results in either increased or decreased levels of ROS. In previous publications, we have demonstrated that one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is to control the mitochondrial redox balance, and thereby mediate the cellular defense against oxidative damage, via the production of NADPH. To explore the association between IDH2 expression and cardiac function, we measured myocardial hypertrophy, apoptosis, and contractile dysfunction in IDH2 knockout (idh2^−/−) and wild-type (idh2^+/+) mice. As expected, mitochondria from the hearts of knockout mice lacked IDH2 activity and the hearts of IDH2-deficient mice developed accelerated heart failure, increased levels of apoptosis and hypertrophy, and exhibited mitochondrial dysfunction, which was associated with a loss of redox homeostasis. Our results suggest that IDH2 plays an important role in maintaining both baseline mitochondrial function and cardiac contractile function following pressure-overload hypertrophy, by preventing oxidative stress.     

Loss of lipoprotein lipase-derived fatty acids leads to increased cardiac glucose metabolism and heart dysfunction

Long-chain fatty acids (FAs) are the predominant energy substrate utilized by the adult heart. The heart can utilize unesterified FA bound to albumin or FA obtained from lipolysis of lipoprotein-bound triglyceride (TG). We used heart-specific lipoprotein lipase knock-out mice (hLpL0) to test whether these two sources of FA are interchangeable and necessary for optimal heart function. Hearts unable to obtain FA from lipoprotein TG were able to compensate by increasing glucose uptake, glycolysis, and glucose oxidation. HLpL0 hearts had decreased expression of pyruvate dehydrogenase kinase 4 and increased cardiomyocyte expression of glucose transporter 4. Conversely, FA oxidation rates were reduced in isolated perfused hLpL0 hearts. Following abdominal aortic constriction expression levels of genes regulating FA and glucose metabolism were acutely up-regulated in control and hLpL0 mice, yet all hLpL0 mice died within 48 h of abdominal aortic constriction. Older hLpL0 mice developed cardiac dysfunction characterized by decreased fractional shortening and interstitial and perivascular fibrosis. HLpL0 hearts had increased expression of several genes associated with transforming growth factor-beta signaling. Thus, long term reduction of lipoprotein FA uptake is associated with impaired cardiac function despite a compensatory increase in glucose utilization.