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The Ras family of small Guanosine Triphosphate (GTP)-binding proteins (G proteins) represents one of the main components of intracellular signal transduction required for normal cardiac growth, but is also critically involved in the development of cardiac hypertrophy and heart failure. The present review provides an update on the role of the H-, K- and N-Ras genes and their related pathways in cardiac diseases. We focus on cardiac hypertrophy and heart failure, where Ras has been studied the most. We also review other cardiac diseases, like genetic disorders related to Ras. The scope of the review extends from fundamental concepts to therapeutic applications. Although the three Ras genes have a nearly identical primary structure, there are important functional differences between them: H-Ras mainly regulates cardiomyocyte size, whereas K-Ras regulates cardiomyocyte proliferation. N-Ras is the least studied in cardiac cells and is less associated to cardiac defects. Clinically, oncogenic H-Ras causes Costello syndrome and facio-cutaneous-skeletal syndromes with hypertrophic cardiomyopathy and arrhythmias. On the other hand, oncogenic K-Ras and alterations of other genes of the Ras-Mitogen-Activated Protein Kinase (MAPK) pathway, like Raf, cause Noonan syndrome and cardio-facio-cutaneous syndromes characterized by cardiac hypertrophy and septal defects. We further review the modulation by Ras of key signaling pathways in the cardiomyocyte, including: (i) the classical Ras-Raf-MAPK pathway, which leads to a more physiological form of cardiac hypertrophy; as well as other pathways associated with pathological cardiac hypertrophy, like (ii) The SAPK (stress activated protein kinase) pathways p38 and JNK; and (iii) The alternative pathway Raf-Calcineurin-Nuclear Factor of Activated T cells (NFAT). Genetic alterations of Ras isoforms or of genes in the Ras-MAPK pathway result in Ras-opathies, conditions frequently associated with cardiac hypertrophy or septal defects among other cardiac diseases. Several studies underline the potential role of H- and K-Ras as a hinge between physiological and pathological cardiac hypertrophy, and as potential therapeutic targets in cardiac hypertrophy and failure. Graphic abstract, Highlights The Ras (Rat Sarcoma) gene family is a group of small G proteins Ras is regulated by growth factors and neurohormones affecting cardiomyocyte growth and hypertrophy Ras directly affects cardiomyocyte physiological and pathological hypertrophy Genetic alterations of Ras and its pathways result in various cardiac phenotypes Ras and its pathway are differentially regulated in acquired heart disease Ras modulation is a promising therapeutic target in various cardiac conditions.

Programmed cell death receptor-1 blocking antibodies and cytotoxic T-lymphocyte–associated antigen-4 blocking antibodies are referred to as checkpoint inhibitors and are used in cancer immunotherapy. While enhancing T-cell antitumor immunity, checkpoint inhibitors can also cause immune-mediated nephrotoxicities, manifesting mostly in acute kidney injury. Cytotoxic T-lymphocyte–associated antigen-4 blockade was associated with acute interstitial nephritis with variable degrees of podocyte effacement, minimal change disease, and membranous nephropathy. Conversely, programmed cell death receptor-1 blockade has mostly been associated with acute interstitial nephritis, with or without various glomerular diseases. In particular, cases of minimal change disease were reported with programmed cell death receptor-1 blockade, including two cases of minimal change disease reported in Hodgkin’s lymphoma and one with mesothelioma, in addition to one case of focal segmental glomerulosclerosis after treatment of renal cell carcinoma. We report a case of acute interstitial nephritis and minimal change disease in a patient with Hodgkin’s lymphoma treated with nivolumab and discuss pathophysiological hypotheses. The association of minimal change disease with Hodgkin’s lymphoma is well-recognized and may be related to c-mip protein and/or CD80. The checkpoint pathway likely triggers the auto-immune, T-cell-mediated, minimal change disease associated with Hodgkin’s lymphoma; however, this pathogenic process may also involve a programmed cell death receptor-1– fyn-c-mip interaction or a PD-L1–CD80 cross-talk at both T-cell and podocyte level.

Intracellular calcium (Ca2+) is a critical coordinator of various aspects of cellular physiology. It is increasingly apparent that changes in cellular Ca2+ dynamics contribute to the regulation of normal and pathological signal transduction that controls cell growth and survival. Aberrant perturbations in Ca2+ homeostasis have been implicated in a range of pathological conditions, such as cardiovascular diseases, diabetes, tumorigenesis and steatosis hepatitis. Intracellular Ca2+ concentrations are therefore tightly regulated by a number of Ca2+ handling enzymes, proteins, channels and transporters located in the plasma membrane and in Ca2+ storage organelles, which work in concert to fine tune a temporally and spatially precise Ca2+ signal. Chief amongst them is the sarco/endoplasmic reticulum (SR/ER) Ca2+ ATPase pump (SERCA) which actively re-accumulates released Ca2+ back into the SR/ER, therefore maintaining Ca2+ homeostasis. There are at least 14 different SERCA isoforms encoded by three ATP2A1-3 genes whose expressions are species- and tissue-specific. Altered SERCA expression and activity results in cellular malignancy and induction of ER stress and ER stress-associated apoptosis. The role of SERCA misregulation in the control of apoptosis in various cell types and disease setting with prospective therapeutic implications is the focus of this review. Ca2+ is a double edge sword for both life as well as death, and current experimental evidence supports a model in which Ca2+ homeostasis and SERCA activity represent a nodal point that controls cell survival. Pharmacological or genetic targeting of this axis constitutes an incredible therapeutic potential to treat different diseases sharing similar biological disorders.

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 β 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.

Background Clinical two-dimensional (2D) and clinical three-dimensional echocardiography are validated against cardiac magnetic resonance imaging (CMR), the gold standard for left ventricular (LV) volume measurement. In rodents, there is no widely accepted echocardiographic measure of whole LV volumes, and CMR measurements vary among studies. The aim of this study was to compare LV volumes by 2D echocardiography (using a hemisphere-cylinder [HC] model) with HC and full-volume (FV) CMR in normal and diseased rats to measure the impact of geometric models and imaging modalities. Methods Rats ( n = 27) underwent ascending aortic banding, myocardial infarction induction by either permanent left anterior descending coronary artery ligation or ischemia-reperfusion, and sham thoracotomy. Subsequently, end-diastolic volume, end-systolic volume, and ejection fraction were measured using an HC 2D echocardiographic model combining parasternal short-axis and long-axis measurements, and these were compared with HC and FV CMR. Results Diseased groups showed LV dilatation and dysfunction. HC echocardiographic and FV CMR measures of end-diastolic volume, end-systolic volume, and ejection fraction were correlated. On Bland-Altman plots, end-diastolic volumes were concordant between both methods, while HC echocardiography underestimated end-systolic volumes, resulting in a modest overestimation of ejection fractions compared with FV CMR. Other 2D echocardiographic geometric models offered less concordance with FV CMR than HC. HC CMR overestimated LV volumes compared with FV CMR, while HC echocardiography underestimated HC CMR volumes. Echocardiography underestimated corresponding LV dimensions by CMR, particularly short axis. Conclusions Concordant measures of LV volume and function were obtained using (1) a relatively simple HC model of the left ventricle inclusive of two orthogonal 2D echocardiographic planes and (2) FV CMR in normal and diseased rats. The HC model appeared to compensate for the underestimation of LV dimensions by echocardiography.

Among the various cardiac contractility parameters, left ventricular (LV) ejection fraction (EF) and maximum dP/d t (dP/d tmax) are the simplest and most used. However, these parameters are often reported together, and it is not clear if they are complementary or redundant. We sought to compare the discriminative value of EF and dP/d tmax in assessing systolic dysfunction after myocardial infarction (MI) in swine. A total of 220 measurements were obtained. All measurements included LV volumes and EF analysis by left ventriculography, invasive ventricular pressure tracings, and echocardiography. Baseline measurements were performed in 132 pigs, and 88 measurements were obtained at different time points after MI creation. Receiver operator characteristic (ROC) curves to distinguish the presence or absence of an MI revealed a good predictive value for EF [area under the curve (AUC): 0.998] but not by dP/d tmax (AUC: 0.69, P < 0.001 vs. EF). Dividing dP/d tmax by LV end-diastolic pressure and heart rate (HR) significantly increased the AUC to 0.87 ( P < 0.001 vs. dP/d tmax and P < 0.001 vs. EF). In naïve pigs, the coefficient of variation of dP/d tmax was twice than that of EF (22.5% vs. 9.5%, respectively). Furthermore, in n = 19 pigs, dP/d tmax increased after MI. However, echocardiographic strain analysis of 23 pigs with EF ranging only from 36% to 40% after MI revealed significant correlations between dP/d tmax and strain parameters in the noninfarcted area (circumferential strain: r = 0.42, P = 0.05; radial strain: r = 0.71, P < 0.001). In conclusion, EF is a more accurate measure of systolic dysfunction than dP/d tmax in a swine model of MI. Despite the variability of dP/d tmax both in naïve pigs and after MI, it may sensitively reflect the small changes of myocardial contractility.

Use of gene therapy for heart failure is gaining momentum as a result of the recent successful completion of phase II of the Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial, which showed clinical safety and efficacy of an adeno-associated viral vector expressing sarco-endoplasmic reticulum calcium ATPase (SERCA2a). Resorting to gene therapy allows the manipulation of molecular targets not presently amenable to pharmacologic modulation. This short review focuses on the molecular targets of heart failure gene therapy that have demonstrated translational potential. At present, most of these targets are related to calcium handling in the cardiomyocyte. They include SERCA2a, phospholamban, S100A1, ryanodine receptor, and the inhibitor of the protein phosphatase 1. Other targets related to cAMP signaling are reviewed, such as adenylyl cyclase. MicroRNAs are emerging as novel therapeutic targets and convenient vectors for gene therapy, particularly in heart disease. We propose a discussion of recent advances and controversies in key molecular targets of heart failure gene therapy.

We have previously reported that resistin induces hypertrophy and impairs contractility in isolated rat cardiomyocytes. To examine the long-term cardiovascular effects of resistin, we induced in vivo overexpression of resistin using adeno-associated virus serotype 9 injected by tail vein in rats and compared to control animals. Ten weeks after viral injection, overexpression of resistin was associated with increased ratio of left ventricular (LV) weight/body weight, increased end-systolic LV volume and significant decrease in LV contractility, measured by the end-systolic pressure volume relationship slope in LV pressure volume loops, compared to controls. At the molecular level, mRNA expression of ANF and β-MHC, and protein levels of phospholamban were increased in the resistin group without a change in the level of SERCA2a protein expression. Increased fibrosis by histology, associated with increased mRNA levels of collagen, fibronectin and connective tissue growth factor were observed in the resistin-overexpressing hearts. Resistin overexpression was also associated with increased apoptosis in vivo, along with an apoptotic molecular phenotype in vivo and in vitro. Resistin-overexpressing LV tissue had higher levels of TNF-α receptor 1 and iNOS, and reduced levels of eNOS. Cardiomyocytes overexpressing resistin in vitro produced larger amounts of TNFα in the medium, had increased phosphorylation of IκBα and displayed increased intracellular reactive oxygen species (ROS) content with increased expression and activity of ROS-producing NADPH oxidases compared to controls. Long-term resistin overexpression is associated with a complex phenotype of oxidative stress, inflammation, fibrosis, apoptosis and myocardial remodeling and dysfunction in rats. This phenotype recapitulates key features of diabetic cardiomyopathy. This article is part of Special Issue Item Group entitled "Possible Editorial".

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 β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-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.

Chronically elevated levels of oxidative stress resulting from increased production and/or impaired scavenging of reactive oxygen species are a hallmark of mitochondrial dysfunction in left ventricular hypertrophy. Recently, oscillations of the mitochondrial membrane potential (DeltaPsi(m)) were mechanistically linked to changes in cellular excitability under conditions of acute oxidative stress produced by laser-induced photooxidation of cardiac myocytes in vitro. Here, we investigate the spatiotemporal dynamics of DeltaPsi(m) within the intact heart during ischemia-reperfusion injury. We hypothesize that altered metabolic properties in left ventricular hypertrophy modulate DeltaPsi(m) spatiotemporal properties and arrhythmia propensity.

Acute coronary occlusion is the leading cause of death in the Western world. There is an unmet need for the development of treatments to limit the extent of myocardial infarction (MI) during the acute phase of occlusion. Recently, investigators have focused on the use of a chemokine, CXCL12, the only identified ligand for CXCR4, as a new therapeutic modality to recruit stem cells to individuals suffering from MI. Here, we examined the effects of overexpression of CXCR4 by gene transfer on MI. Adenoviruses carrying the CXCR4 gene were injected into the rat heart one week before ligation of the left anterior descending coronary artery followed by 24 hours reperfusion. Cardiac function was assessed by echocardiography couple with 2,3,5-Triphenyltetrazolium chloride staining to measure MI size. In comparison with control groups, rats receiving Ad-CXCR4 displayed an increase in infarct area (13.5% +/- 4.1%) and decreased fractional shortening (38% +/- 5%). Histological analysis revealed a significant increase in CXCL12 and tumor necrosis factor-alpha expression in ischemic area of CXCR4 overexpressed hearts. CXCR4 overexpression was associated with increased influx of inflammatory cells and enhanced cardiomyocyte apoptosis in the infarcted heart. These data suggest that in our model overexpressing CXCR4 appears to enhance ischemia/reperfusion injury possibly due to enhanced recruitment of inflammatory cells, increased tumor necrosis factor-alpha production, and activation of cell death/apoptotic pathways.

The cardiac isoform of the sarco/endoplasmic reticulum Ca(2+)ATPase (SERCA2a) plays a major role in controlling excitation/contraction coupling. In both experimental and clinical heart failure, SERCA2a expression is significantly reduced which leads to abnormal Ca(2+) handling and deficient contractility. A large number of studies in isolated cardiac myocytes and in small and large animal models of heart failure showed that restoring SERCA2a expression by gene transfer corrects the contractile abnormalities and improves energetics and electrical remodeling. Following a long line of investigation, a clinical trial is underway to restore SERCA2a expression in patients with heart failure using adeno-associated virus type 1. This review addresses the following issues regarding heart failure gene therapy: i) new insights on calcium regulation by SERCA2a; ii) SERCA2a as a gene therapy target in animal models of heart failure; iii) advances in the development of viral vectors and gene delivery; and iv) clinical trials on heart failure using SERCA2a. This review focuses on the new advances in SERCA2a- targeted gene therapy made in the last three years. In conclusion, SERCA2a is an important therapeutic target in various cardiovascular disorders. Ongoing clinical gene therapy trials will provide answers on its safety and applicability.

Eccentric cardiac remodeling seen in dilated cardiomyopathy or regurgitant valvular disease is a well-known process of heart failure progression, but its mechanoenergetic profile has not been yet established. We made a volume overload (VO) heart failure model in rats and for the first time investigated left ventricular (LV) mechanical work and energetics in cross-circulated whole heart preparations. Laparotomy was performed in 14 Wistar male rats, and abdominal aortic-inferior vena caval shunt was created in seven rats (VO group). Another seven rats underwent a sham operation without functional shunt (Sham group). LV dimensions changes were followed with weekly transthoracic echocardiography. Three months after surgery, we measured LV pressure and volume and myocardial O2 consumption in isolated heart cross circulation. LV internal dimensions in both systolic and diastolic phases were significantly increased in the VO group versus the Sham group ( P < 0.05). LV pressure was markedly decreased in the VO group versus in the Sham group ( P < 0.05). LV end-systolic pressure-volume relation shifted downward, and myocardial O2 consumption related to Ca2+ handling significantly decreased. The contractile response to Ca2+ infusion was attenuated. Nevertheless, the increase in Ca2+ handling-related O2 consumption per unit change in LV contractility in the VO group was significantly higher than that in the Sham group ( P < 0.05). The levels of sarco(endo)plasmic reticulum Ca2+-ATPase 2a protein were reduced in the VO group ( P < 0.01). In conclusion, VO failing rat hearts had a character of marked contractile dysfunction accompanied with less efficient energy utilization in the Ca2+ handling processes. These results suggest that restoring Ca2+ handling in excitation-contraction coupling would improve the contractility of the myocardium after eccentric cardiac remodeling.

Background— RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. Here, we report on targeted RNAi for the treatment of heart failure, an important disorder in humans that results from multiple causes. Successful treatment of heart failure is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban, a key regulator of cardiac Ca 2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy uses regulatory RNAs to achieve its effect. Methods and Results— We describe structural requirements to obtain high RNAi activity from adenoviral and adeno-associated virus (AAV9) vectors and show that an adenoviral short hairpin RNA vector (AdV-shRNA) silenced phospholamban in cardiomyocytes (primary neonatal rat cardiomyocytes) and improved hemodynamics in heart-failure rats 1 month after aortic root injection. For simplified long-term therapy, we developed a dimeric cardiotropic adeno-associated virus vector (rAAV9-shPLB) to deliver RNAi activity to the heart via intravenous injection. Cardiac phospholamban protein was reduced to 25%, and suppression of sacroplasmic reticulum Ca 2+ ATPase in the HF groups was rescued. In contrast to traditional vectors, rAAV9 showed high affinity for myocardium but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (left ventricular end-diastolic pressure, dp/dt min , and τ) and systolic (fractional shortening) functional parameters to normal ranges. The massive cardiac dilation was normalized, and cardiac hypertrophy, cardiomyocyte diameter, and cardiac fibrosis were reduced significantly. Importantly, no evidence was found of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusions— Our data show for the first time the high efficacy of an RNAi therapeutic strategy in a cardiac disease.

The aim of this study was to examine how global cardiac gene transfer of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) can influence left ventricular (LV) mechanical and energetic function, especially in terms of O2 cost of LV contractility, in normal rats. Normal rats were randomized to receive an adenovirus carrying the SERCA2a (SERCA) or β-galactosidase (β-Gal) gene or saline by a catheter-based technique. LV mechanical and energetic function was measured in cross-circulated heart preparations 2–3 days after the infection. The end-systolic pressure-volume relation was shifted upward, end-systolic pressure at 0.1 ml of intraballoon water volume was higher, and equivalent maximal elastance, i.e., enhanced LV contractility, was higher in the SERCA group than in the normal, β-Gal, and saline groups. Moreover, the LV relaxation rate was faster in the SERCA group. There was no significant difference in myocardial O2 consumption per beat-systolic pressure-volume area relation among the groups. Finally, O2 cost of LV contractility was decreased to subnormal levels in the SERCA group but remained unchanged in the β-Gal and saline groups. This lowered O2 cost of LV contractility in SERCA hearts indicates energy saving in Ca2+ handling during excitation-contraction coupling. Thus overexpression of SERCA2a transformed the normal energy utilization to a more efficient state in Ca2+ handling and superinduced the supranormal contraction/relaxation due to enhanced Ca2+ handling.

Recombinant adeno-associated (AAV) viruses have unique properties, which make them ideal vectors for gene transfer targeting the myocardium. Numerous serotypes of AAV have been identified with variable tropisms towards cardiac tissue. In the present study, we investigated the time course of expression of eight different AAV serotypes in rat myocardium and the nature of the immunity against these serotypes. We first assessed whether neutralizing antibodies (NAb) were present for any of the serotype in the rats. We injected 100 microl of each AAV 1-8 serotype (10(12) DNAse resistant particles/ml), encoding LacZ gene, into the apical wall of rat myocardium. At 1, 4, 12 and 24 weeks after gene delivery, the animals were killed and beta-galactosidase (beta-gal) activity was assessed by luminometry. Additionally, LacZ genomic copies and AAV capsids copies were measured through standard polymerase chain reaction analysis and cryo-sections from the area of viral injection were stained for X-gal detection at the same time points. No NAbs were detected against any of AAV serotypes. At all the time points studied, AAV1, 6 and 8 demonstrated the highest efficiency in transducing rat hearts in vivo. Parallel to the results with beta-gal activity, the highest levels LacZ and AAV DNA genomic copies were with AAV1, 6 and 8. The positive X-gal staining depicted by these serotypes confirmed these results. These results indicate that among the various AAV serotypes, AAV1, 6 and 8 have differential tropism for the heart unaffected by pre-existing NAb in the rat. Although AAV 1 and 6 vectors induced rapid and robust expression and reach a plateau at 4 weeks, AAV 8 continued increasing until the end of the study. AAV 2, 5 and 7 vectors were slower to induce expression of the reporter gene, but did reach levels of expression comparable to AAV1 and AAV6 vectors after 3 months.

Introduction: Agonists of the Adenosine 2a Receptor (A2aR) are used clinically as pharm-stress agents for perfusion imaging, but they are also potent immunomodulators that reduce the size of acute MI when administered at reperfusion in both large and small animal models of MI. Hypothesis: Sustained administration of an A2aR agonist can reduce post-MI LV remodeling and improve cardiac strain even when treatment is withheld until after infarct size has stabilized. Methods: Two groups of mice were studied: C57BL/6 mice treated with Vehicle or a highly selective A2aR agonist (ATL313). All mice received 1h coronary occlusion and 28d of reperfusion. ATL313 was administered for 28 days by subcutaneous micro-osmotic pumps implanted after MI. All mice underwent 7T CMR imaging at baseline and 2, 7 & 28 days post-MI. CMR included short-axis black-blood cines covering the entire heart, with mid-ventricular cine DENSE for circumferential strain (Ecc). Late Gd-enhanced (LGE) inversion recovery imaging was performed on Days 2&7 and molecular imaging with a collagen-targeted Gd contrast agent (EP3533) on Days 14&28. Mice with Day 2 LGE infarct sizes less than 22% or >42% LV mass were excluded from analysis. Results: Examples of LGE and DENSE analysis are shown in Panels A&B. Day 2 infarct size was similar between groups (ATL313 (n=9): 35±2 vs Vehicle (n=8): 34±2, mean±SEM, p=NS). In panel C, ATL313 significantly improved LV end-systolic volume as early as 2 days post-MI (mean±SEM, *p Conclusions: Pharmacologic immunomodulation with an A2aR agonist inhibits LV remodeling, improves contractile function in infarct-adjacent regions at Day 2 post-MI and reduces LV mass at Day 28. Combined with previous work, these results suggest that A2aR stimulation may prove beneficial in both acute and sub-acute MI.

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 2weeks 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.

Background Pulse pressure and aortic stiffness are both predictors of coronary artery disease. Whether these parameters are directly related to coronary structural alterations has never been studied. Methods From September 1999 to September 2000, the following data were collected from 99 eligible patients: invasive intra-aortic systolic and diastolic blood pressures (BP), extent of coronary artery disease, cardiovascular risk factors, and the incidence of angiographically documented restenosis after coronary angioplasty. Results In the study population, independent determinants of aortic pulse pressure were age, gender, aortic mean BP, heart rate, and extent of coronary artery disease (r2=0.57, P < .0001). In univariate analysis, invasive aortic, but not noninvasive brachial, mean pressure (P = .017) and pulse pressure (P = .027) were significantly associated to the extent of coronary artery disease. In a multiple regression analysis, only male gender (P = .013) and the level of aortic pulse pressure (P = .023) were independently associated with the extent of coronary heart disease. Restenosis was angiographically documented in 11 patients (11%). There was a borderline significant association of restenosis to aortic mean BP (P = .05) and to a past history of multiple previous angioplasties (P = .03). Conclusions In this study, aortic pulse pressure was a significant risk factor for the extent of coronary artery disease. There was only a borderline significant association of restenosis to the steady, but not pulsatile, component of aortic BP in the stent era.

International audience; The sarco(endo)plasmic reticulum Ca(2+)ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis). Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (similar to 99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for similar to 91% of total SERCA and SERCA2b for similar to 5%. Among SERCA3, SERCA3b was the most expressed (similar to 3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b. In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively. In conclusion: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is similar to 30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation-contraction coupling in both CMs and contractile VSMCs.

Patients with Marfan syndrome (MFS), a multisystem disorder caused by mutations in the gene encoding the extracellular matrix (ECM) protein fibrillin 1, are unusually vulnerable to stress-induced cardiac dysfunction. The prevailing view is that MFS-associated cardiac dysfunction is the result of aortic and/or valvular disease. Here, we determined that dilated cardiomyopathy (DCM) in fibrillin 1-deficient mice is a primary manifestation resulting from ECM-induced abnormal mechanosignaling by cardiomyocytes. MFS mice displayed spontaneous emergence of an enlarged and dysfunctional heart, altered physical properties of myocardial tissue, and biochemical evidence of chronic mechanical stress, including increased angiotensin II type I receptor (AT1R) signaling and abated focal adhesion kinase (FAK) activity. Partial fibrillin 1 gene inactivation in cardiomyocytes was sufficient to precipitate DCM in otherwise phenotypically normal mice. Consistent with abnormal mechanosignaling, normal cardiac size and function were restored in MFS mice treated with an AT1R antagonist and in MFS mice lacking AT1R or β-arrestin 2, but not in MFS mice treated with an angiotensin-converting enzyme inhibitor or lacking angiotensinogen. Conversely, DCM associated with abnormal AT1R and FAK signaling was the sole abnormality in mice that were haploinsufficient for both fibrillin 1 and β1 integrin. Collectively, these findings implicate fibrillin 1 in the physiological adaptation of cardiac muscle to elevated workload.

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 β 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.

Heart failure (HF) is a vicious circle in which an original insult leading to mechanical cardiac dysfunction initiates multiple morphological, biochemical and molecular pathological alterations referred to as cardiac remodelling. Remodelling leads to further deterioration of cardiac function and functional reserve. Interrupting or reversing cardiac remodelling is a major therapeutic goal of HF therapies. The role of molecules and molecular pathways in cardiac remodelling and HF has been extensively studied. Multiple approaches are now used or investigated in HF therapy, including pharmacological therapy, device therapy, gene therapy, cell therapy and biological therapy targeting cytokines and growth factors. This review explores the molecular targets and molecular bases of current and prospective therapies in HF.

Deregulated or enhanced calcium ion (Ca2+) influx across an unstable sarcolemma has been proposed to directly affect cardiac hypertrophic remodelling, vascular proliferative diseases and degenerative muscle disorders. Aberrant intracellular handling is partly due to a defect in Sarcoplasmic Reticulum (SR) function. Decreased Ca2+ uptake in cardiac, vascular and skeletal myocytes is associated with a decrease in the expression and activity of the fast sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a or SERCA1a isoforms). SERCA2a gene transfer was successfully used in heart failure; this approach holds further therapeutic promises in vascular proliferative diseases and dystrophin-deficient muscular diseases. The growing family of human SERCA isoforms comprises at least 14 mRNA and proteins with different functional characteristics and cell-specific expression. This review focuses on the biological role and therapeutic potential of different isoforms of SERCA in the physiology and pathology of cardiac, vascular and skeletal muscle cells.

Load-adjusted measures of left ventricle (LV) systolic performance are limited by dependence on LV stiffness and afterload. To our knowledge, no stiffness-adjusted and afterload-adjusted indicator was tested in models of pressure (POH) and volume overload hypertrophy (VOH). We hypothesized that wall stress reflects changes in loading, incorporating chamber stiffness and afterload; therefore, stroke volume-to-wall stress ratio more accurately reflects systolic performance. We used rat models of POH (ascending aortic banding) and VOH (aorto-cava shunt). Animals underwent echocardiography and pressure-volume analysis at baseline and dobutamine challenge. We achieved extreme bidirectional alterations in LV systolic performance, end-systolic elastance (Ees), passive stiffness, and arterial elastance (Ea). In POH with LV dilatation and failure, some load-independent indicators of systolic performance remained elevated compared with controls, while some others failed to decrease with wide variability. In VOH, most, but not all indicators, including LV ejection fraction, were significantly reduced compared with controls, despite hyperdynamic circulation, lack of heart failure, and preserved contractile reserve. We related systolic performance to Ees adjusted for Ea and LV passive stiffness in multivariate models. Calculated residual Ees was not reduced in POH with heart failure and was reduced in VOH, while it positively correlated to dobutamine dose. Conversely, stroke volume-to-wall stress ratio was normal in compensated POH, markedly decreased in POH with heart failure, and, in contrast with LV ejection fraction, normal in VOH. Our results support stroke volume-to-wall stress ratio as a load-adjusted and stiffness-adjusted indicator of systolic function in models of POH and VOH.

Aortic pulse pressure (APP) is related to arterial stiffness and associated with the presence and extent of coronary artery disease (CAD). Besides, the left coronary artery (LCA) has a predominantly diastolic flow while the right coronary artery (RCA) receives systolic and diastolic flow. Thus, we hypothesized that increased systolic-diastolic pressure difference had a greater atherogenic effect on the RCA than on the LCA.A random sample of 433 CAD patients (145 females, 288 males, mean age 65.0 ± 11.1 years) undergoing coronary angiography at Staten Island University Hospital between January 2005 and May 2008 was studied. Coronary lesion was defined as a ≥50% luminal stenosis. Patients were divided into three groups, with isolated LCA lesions (n = 154), isolated RCA lesions (n = 36) or mixed LCA and RCA lesions (n = 243).APP differed significantly between groups, being highest when the RCA alone was affected (67.6 ± 20.3 mm Hg for LCA vs. 78.8 ± 22.0 for RCA vs. 72.7 ± 22.6 for mixed, P = 0.008 for analysis of variance (ANOVA)). Age and gender were not associated with CAD location. Heart rate was associated with CAD location, lowest in RCA group, and negatively correlated with APP. However, left ventricular ejection fraction (LVEF) was lower in the mixed CAD group and positively correlated with APP. The association between APP and right-sided CAD persisted in multivariate logistic regression adjusting for confounders, including heart rate, LVEF and medication use. A similar but less significant pattern was seen with brachial arterial pressures.Aortic pulse pressure may affect CAD along with coronary flow phasic patterns.

The aim of this study was to reveal the temporal and spatial changes of strain parameters during the progression of chronic coronary ischemia. Fourteen pigs received occluder implantation to create gradual ischemia (CI), while six pigs underwent a sham surgery (Control). Six pigs after myocardial infarction were also studied (MI). Strain analysis was performed using a speckle-tracking algorithm. Eleven of the 14 animals with occluder implantation had total occlusion of the left anterior descending artery with collaterals at 1 month (early occlusion group), whereas three pigs had occlusion at 3 months (late occlusion group). Both radial strain (RS) and circumferential strain (CS) of ischemic area deteriorated at 1 month in the early occlusion group and remained at the same level throughout the remaining 2 months of the experiment. In the late occlusion group, RS gradually declined, while CS took the same course as Control until the 2 month time point. Thereafter, both metrics reached the same level as the early occlusion group at the time of occlusion. Interestingly, RS in the remote area decreased moderately, whereas CS remained normal in CI pigs. The comparison between CI and MI revealed preserved CS at the ischemic area in CI pigs. Both RS and CS deteriorate by the time total coronary occlusion was established and remain at the same level thereafter. Altered RS in the remote area may be an indicator of remodeling in the non-ischemic area, whereas CS may be useful for distinguishing between transmural and non-transmural scar.

Current rodent models of ischemia/infarct or pressure-volume overload are not fully representative of human heart failure. We developed a new model of congestive heart failure (CHF) with both ischemic and stress injuries combined with fibrosis in the remote myocardium. Sprague-Dawley male rats were used. Ascending aortic banding (Ab) was performed to induce hypertrophy. Two months post-Ab, ischemia-reperfusion (I/R) injury was induced by ligating the left anterior descending (LAD) artery for 30 min. Permanent LAD ligation served as positive controls. A debanding (DeAb) procedure was performed after Ab or Ab + I/R to restore left ventricular (LV) loading properties. Cardiac function was assessed by echocardiography and in vivo hemodynamic analysis. Myocardial infarction (MI) size and myocardial fibrosis were assessed. LV hypertrophy was observed 4 mo post-Ab; however, systolic function was preserved. LV hypertrophy regressed within 1 mo after DeAb. I/R for 2 mo induced a small to moderate MI with mild impairment of LV function. Permanent LAD ligation for 2 mo induced large MI and significant cardiac dysfunction. Ab for 2 mo followed by I/R for 2 mo (Ab + I/R) resulted in moderate MI with significantly reduced ejection fraction (EF). DeAb post Ab + I/R to reduce afterload could not restore cardiac function. Perivascular fibrosis in remote myocardium after Ab + I/R + DeAb was associated with decreased cardiac function. We conclude that Ab plus I/R injury with aortic DeAb represents a novel model of CHF with increased fibrosis in remote myocardium. This model will allow the investigation of vascular and fibrotic mechanisms in CHF characterized by low EF, dilated LV, moderate infarction, near-normal aortic diameter, and reperfused coronary arteries.

Background/Aim Diabetes-associated myocardial dysfunction results in altered gene expression in the heart. We aimed to investigate the changes in gene expression profiles accompanying diabetes-induced cardiomyopathy and its phenotypic rescue by restoration of SERCA2a expression. Methods/Results Using the Otsuka Long-Evans Tokushima Fatty rat model of type 2 diabetes and the Agilent rat microarray chip, we analyzed gene expression by comparing differential transcriptional changes in age-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a. Microarray expression profiles of selected genes were verified with real-time qPCR and immunoblotting. Our analysis indicates that diabetic cardiomyopathy is associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. SERCA2a gene transfer in these hearts reduced diabetes-associated hypertrophy, and differentially modulated the expression of 76 genes and reversed the transcriptional profile induced by diabetes. In isolated cardiomyocytes in vitro, SERCA2a overexpression significantly modified the expression of a number of transcripts known to be involved in insulin signaling, glucose metabolism and cardiac remodeling. Conclusion This investigation provided insight into the pathophysiology of cardiac remodeling and the potential role of SERCA2a normalization in multiple pathways in diabetic cardiomyopathy.

Increased production and/or impaired scavenging of reactive oxygen species (ROS) are a hallmark of mitochondrial dysfunction in structural heart disease. Previously, we demonstrated a mechanism by which depolarization of the mitochondrial membrane potential (mitoVm) caused by ROS release through the mitochondrial benzodiazepine receptor (mBZR) promotes conduction failure. Since pressure overload hypertrophy (PoH) is a major risk factor for sudden death, we hypothesized that instability of mitoVm in PoH predisposes to arrhythmias. Methods: PoH was produced in rats by ascending aortic banding for 4 weeks. LV dysfunction characteristic of PoH was confirmed in vivo using M mode 2D echocardiography. Ultrahigh resolution optical imaging was performed in ex vivo perfused hearts from PoH (n=6) and normal (n=3) rats. Normalized mitoVm was measured using a novel quantitative technique of TMRM imaging with subcellular resolution during a protocol of ischemia (for 7min) and reperfusion. Mean and SD mitoVm across 6400 sites were compared as was the expression of the mBZR gene (PKBS) using western blot analysis. Results: Aortic banding for 4 weeks resulted in a significant increase in LV anteroseptal and posterior wall dimensions consistent with structural remodeling in PoH. Ischemia reperfusion related ventricular fibrillation (VF) was observed in 5/6 PoH and 1/3 normal hearts. Average mitoVm was surprisingly greater in PoH compared to normal hearts during both ischemia (17%, p=0.012) and reperfusion (24%, p=0.004). However, spatial heterogeneity of mitoVm indexed by SD was markedly greater in PoH during ischemia (0.42 vs 0.11, p=0.00007) but not reperfusion (p=0.15). Analysis of mitoVm in susceptible (VF+) versus resistant (VF−) rats revealed a 4X increase in mitoVm heterogeneity in ischemia (p=0.0004) but not reperfusion (p=0.12) in the VF+ group. There were no differences in overall protein content of PKBS between groups indicating that altered mitoVm dynamics are independent of PKBS expression. Conclusions: These data demonstrate that increased spatial heterogeneity of mitoVm during ischemia in PoH promotes VF. Strategies aimed at preventing the desynchronization of mitoVm may form a novel therapeutic approach for combating malignant arrhythmias. This research has received full or partial funding support from the American Heart Association, AHA National Center.

1. The aim of the present study was to examine the acute and chronic effects of adenoviral gene transfer on cardiac function in terms of left ventricular (LV) mechanoenergetic function. Recombinant adenoviral vector carrying beta-galactosidase and green fluorescent protein genes (Ad.betagal-GFP) was used. Cardiac function was examined in cross-circulated rat heart preparations, where end-systolic/diastolic pressure-volume relationships (ESPVR/EDPVR), systolic pressure-volume area (PVA), LV relaxation rate, equivalent maximal elastance at mid-range LV volume (eE(max) at mLVV), coronary blood flow, coronary vascular resistance and myocardial oxygen consumption (VO(2)) were also measured. 2. To examine the ex vivo acute effects of the adenoviral vector, data were obtained before and 30-90 min after intracoronary infusion of Ad.betagal-GFP in the excised, cross-circulated hearts that underwent serotonin pretreatment. To examine the in vivo chronic effects of adenoviral gene transfer, normal rat hearts received Ad.betagal-GFP or saline by a catheter-based technique and data were obtained 3 days after the injection of Ad.betagal-GFP or saline. 3. The ESPVR, EDPVR, LV relaxation rate, eE(max) at mLVV, coronary blood flow and coronary vascular resistance remained unchanged in Ad.betagal-GFP-transfected hearts in both ex vivo acute and in vivo chronic experiments. Moreover, the ex vivo and in vivo transfection caused no change in the slope and VO(2) intercept of the VO(2)-PVA relationship, VO(2) for basal metabolism and for Ca(2+) handling in excitation-contraction coupling and O(2) costs of LV contractility. 4. These results indicate that adenoviral gene transfer has neither acute nor chronic toxic effects on LV mechanical and energetic function. A special combination of in vivo adenoviral gene transfer and a cross-circulation experimental system may provide a useful novel strategy to explore the functional and mechanoenergetic role of specifically targeted genes in the diseased heart.

The aim of this study was to examine whether short- and long-term gene transfer of Ca 2+ handling proteins restore left ventricular (LV) mechanoenergetics in aortic banding-induced failing hearts. Aortic-banded rats received recombinant adenoviruses carrying sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) (Banding + SERCA), parvalbumin (Banding + Parv) or β-galactosidase (Banding + βgal), or an adeno-associated virus carrying SERCA2a (Banding + AAV.SERCA) by a catheter-based technique. LV mechanoenergetic function was measured in cross-circulated hearts. “Banding”, “Banding + βgal” and “Banding + saline” groups showed lower end-systolic pressure at 0.1 ml intraballoon water (ESP 0.1 ), higher end-diastolic pressure at 0.1 ml intraballoon water (EDP 0.1 ) and slower LV relaxation rate, compared with “Normal” and “Sham”. However, “Banding + SERCA” and “Banding + Parv” showed high ESP 0.1 , low EDP 0.1 and fast LV relaxation rate. In “Banding”, “Banding + βgal” and “Banding + saline”, slope of relation between cardiac oxygen consumption and systolic pressure–volume area, O 2 cost of total mechanical energy, was twice higher than normal value, whereas slope in “Baning + SERCA” and “Banding + Parv” was similar to normal value. Furthermore, O 2 cost of LV contractility in the 3 control banding groups was ∼ 3 times higher than normal value, whereas O 2 cost of contractility in “Banding + SERCA”, “Banding + AAV.SERCA” and “Banding + Parv” was as low as normal value. Thus, high O 2 costs of total mechanical energy and of LV contractility in failing hearts indicate energy wasting both in chemomechanical energy transduction and in calcium handling. Improved calcium handling by both short- and long-term overexpression of SERCA2a and parvalbumin transforms the inefficient energy utilization into a more efficient state. Therefore enhancement of calcium handling either by resequestration into the SR or by intracellular buffering improves not only mechanical but energetic function in failing hearts.

Stem cells have been targeted to many organ systems specifically to replace scarred organs and to rejuvenate diseased organs. Even though our understanding of the versatility of stem cells is slowly unraveling, tracking these cells as they enter the body has become a very important field of study. In this chapter, we review various modalities for imaging stem cells and assess the advantages and shortcomings of each technique.

The Otsuka Long-Evans Tokushima fatty rat is an animal model of Type 2 diabetes mellitus (DM), which is characterized by diastolic dysfunction associated with decreased sarcoplasmic reticulum Ca2+-ATPase (SERCA2a). The aim of this study was to examine whether gene transfer of SERCA2a can influence coronary blood flow and cardiomyocyte diameter in this model. DM rats were injected with adenovirus carrying SERCA2a (DM+SERCA) or β-galactosidase gene (DM+βGal). Coronary blood flow was measured in cross-circulated excised hearts 3 days after infection. Although in all groups coronary blood flow remained unchanged even if left ventricular (LV) volume or intracoronary Ca2+ infusion was increased, the DM+SERCA group showed a sustained increase in coronary blood flow compared with the other groups. This result suggests that the sustained high coronary blood flow is a specific response in SERCA2a-overexpressed hearts. Although the LV weight-to-body weight ratio (LV/BW) and cardiomyocyte diameter were higher in the DM and DM+βGal groups than in the non-DM group, in the DM+SERCA group, these measurements were restored to non-DM size. The percentages of collagen area in the three DM groups was significantly higher than results shown in non-DM rats, and there were no significant differences in collagen area percentage among the three DM groups. These results suggest that a lowered LV/BW by SERCA2a overexpression is due mainly to reduced size of cardiomyocytes without any changes in collagen area percentage. In conclusion, in DM failing hearts, SERCA2a gene transfer can increase coronary blood flow and reduce cardiomyocyte size without reduction in collagen production.

The Otsuka-Long-Evans Tokushima Fatty rat represents a model for spontaneous non-insulin-dependent type II diabetes mellitus (DM), characterized by diastolic dysfunction and associated with abnormal calcium handling and decrease in sarcoplasmic reticulum Ca2+ -ATPase (SERCA2a) expression. The aim of this study was to examine whether SERCA2a gene transfer can restore the energetic deficiency and left ventricular (LV) function in this model. DM rats were randomized to receive adenovirus carrying either the SERCA2a gene (DM + Ad.SERCA2a) or the beta-galactosidase gene (DM + Ad.betaGal) or saline (DM + saline). LV mechanoenergetic function was measured in cross-circulated heart preparations 3 days after infection. In DM, end-systolic pressure at 0.1 ml intraballoon water (ESP0.1) was low and end-diastolic pressure at 0.1 ml intraballoon water (EDP0.1) was high (22 mm Hg), compared with non-DM (EDP0.1 12 mm Hg). In DM + Ad.SERCA2a, however, ESP0.1 was increased over 200 mm Hg and EDP(0.1) was decreased to 7 mm Hg. LV relaxation rate was fast in DM + Ad.SERCA2a, but slow in the other DM groups. There was no difference in relation between cardiac oxygen consumption per beat and systolic pressure-volume area among all groups. Finally, the oxygen cost of LV contractility in DM was about three times as high as that of normal. In DM + Ad.SERCA2a, the oxygen cost decreased to control levels, but in DM + Ad.betaGal/DM + saline it remained high. In DM failing hearts, the high oxygen cost indicates energy wasting, which contributes to the contractile dysfunction observed in diabetic cardiomyopathy. SERCA2a gene transfer transforms this inefficient energy utilization into a more efficient state and restores systolic and diastolic function to normal.

Stem cells are a promising approach to cardiovascular therapeutics. Animal experiments have assessed the fate of injected stem cells through ex vivo methods on sacrificed animals. Approaches are needed for in vivo tracking of stem cells. Various imaging techniques and contrast agents for stem cell tracking will be reviewed.

The sarco(endo) plasmic reticulum Ca2+ ATPases (SERCA) system is a key regulator of calcium cycling and signaling in cardiac and vascular myocytes and is composed of several isoforms. We aimed to characterize 1) the expression of the different SERCA isoforms in normal mouse cardiovascular tissues and 2) the modulation of the SERCA isoform system during cardiovascular pathologies (heart failure and/or atherosclerosis). Five SERCA isoforms - 2a, 2b, 3a, 3b and 3c were detected by RT-PCR and immunoblot in normal mouse heart and thoracic aorta. Absolute mRNA quantification (Table) confirmed that SERCA2a was the predominant isoform in the heart (∼99% of total SERCA mRNA) whereas the ubiquitous isoform SERCA2b represents less than ∼0.1%. Within cardiomyocytes (CM), SERCA2a and 2b were co-localized in the longitudinal SR, whereas SERCA3b was located at the junctional reticulum. In the aorta, SERCA2a accounted for ∼86.0% and SERCA2b for ∼7%. Among SERCA3 isoforms (3a, 3b and 3c), SERCA3b was the most expressed (∼6.5%) and was predominantly found in endothelial and vascular smooth muscle cells (VSMC), together with SERCA2a and 2b. In failing CM the SERCA2a expression was 2-fold down-regulated (from 9.65 to 5.01 pg/10 ng of total mRNA). Furthermore, both SERCA2a and SERCA2b were re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels (from 0.27 to 0.13 pg/10 ng of total mRNA) containing mainly synthetic VSMC; the proportion of both SERCA2b and SERCA3b increased to reach 8% and 7% respectively. In summary, we established that 1) the overall expression of SERCA mRNA is ∼ 50 fold higher in the heart compared to vascular tissues; 2) although several SERCA isoforms are expressed in cardiovascular tissues, SERCA2a is the major isoform in both cardiac and vascular myocytes; 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues. Together with the evidence of SERCA2a delocalization from longitudinal reticulum in hypertrophied cardiomyocytes, our study prove that SERCA2a is the principal regulator of excitation-contraction coupling in both CMs and contractile VSMCs and also supports a dynamic and integrated regulation of the SERCA isoform system, which deserves further studies.

Bone marrow mesenchymal stem cells (MSCs) have been used for myocardial regeneration. The fate of injected cells and mechanism of their therapeutic benefits are unclear and cardiomyocytic differentiation is limited. We examined the gene expression of autologous MSCs injected into pig myocardium to assess the biologicprocesses involved.

Bcl-2 E1B 19-KDa interacting protein 3 (BNIP3) is a mitochondrial death and mitophagy marker, which is involved in inducing cardiac remodeling post myocardial infarction. In this study, we show that BNIP3 expression increases in stressed cardiomyocytes in vitro and in response to pressure overload in vivo, and that its transcription is directly related to JNK activity. BNIP3 expression gradually increased in the first weeks after pressure overload and peaked at the heart failure stage. Ultrastructurally, the mitochondrial area was inversely proportional to BNIP3 expression. Both JNK and AKT activities increased with pressure overload; however, JNK signaling dominated over AKT signaling for the activation of the transcription factor FOXO3a and for the transcription of its effector, BNIP3. 3-methyladenine attenuated JNK signaling and significantly decreased BNIP3 expression and reversed cardiac remodeling in heart failure. Ultrastructurally, the mitochondrial area was significantly increased in the 3-methyladenine group compared with placebo. Moreover, adenoviral gene delivery of dominant negative JNK in a rat model of pressure overload hypertrophy abolished the increase in BNIP3 expression in response to pressure overload. These results suggest that JNK signaling is a critical modulator of the transcription factor FOXO3a driving the expression of its effector, BNIP3, in heart failure and that JNK, through BNIP3, induces mitochondrial apoptosis and mitophagy.

Diabetes is associated with impaired cardiac function in both humans and animals. As a consequence of myocardial dysfunction, the expression of several genes within the heart is altered. Our aim is to investigate the changes in gene expression profiles accompanying diabetic cardiomyopathy and to identify molecular and cellular pathways and genes that may contribute to cardiac remodeling as a result of the disease and/or its phenotypic rescue by restoration of the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) through adenoviral gene transfer. Using Otsuka Long-Evans Tokushima Fatty (OLETF) rat model of type II diabetes and the Agilent rat chip, which contains oligonucleotide probe sets for 22,000 rat genes, we performed analyses of gene expression by comparing differential changes in age and sex-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a (which we previously showed to induce functional improvement in heart failure). Microarray results of selected genes were verified with quantitative real-time PCR and immunoblotting. After normalization and filtration of the data and using a 2-fold cut- off, we found that diabetes has differentially induced the expression of 743 genes (216 up- and 527 down-regulated) which take part in many cellular processes. Our analysis indicates that diabetic cardiomyopathy appears to be, in general, associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. Adenoviral gene transfer targeted to these hearts differentially induced the expression of 76 genes and appears, in general, to reverse the transcriptional profile induced by diabetes. Functional gene ontology classification of these genes indicated that SERCA2a restoration is associated with global changes in the cytoskeleton, in cellular energetics and metabolism, in calcium cycling, and in intracellular signaling and transcription regulation pathways. There are also 17 genes of unknown function that are specifically regulated by SERCA overexpression in diabetic hearts. The biological role of some of these novel genes are currently being pursued in other projects. Altogether this investigation provided valuable insight into the pathophysiology of cardiac remodeling and the role of SERCA2a normalization of multiple pathways in diabetes.

Background: The Otsuka-Long-Evans Tokushima Fatty (OLETF) rats represent an unique model of spontaneous non-insulin-dependent diabetes mellitus. From a cardiac standpoint they have been shown to have diastolic dysfunction associated with abnormal calcium handling and a decrease in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) expression. The aim of this study was to examine whether gene transfer of SERCA2a can restore left ventricular (LV) function in this diabetic model in terms of left ventricular (LV) mechanical work and energetics, and the economy of total calcium handling in excitation-contraction (E-C) coupling.