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Background— Magnetic resonance imaging (MRI) in the CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trial revealed that cardiosphere-derived cells (CDCs) decrease scar size and increase viable myocardium after myocardial infarction (MI), but MRI has not been validated as an index of regeneration after cell therapy. We tested the validity of contrast-enhanced MRI in quantifying scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. Methods and Results— Yucatan minipigs underwent induction of MI and 2–3 weeks later were randomized to receive intracoronary infusion of 12.5×10 6 mismatched allogeneic CDCs or vehicle. Allogeneic CDCs induced mild local mononuclear infiltration but no systemic immunogenicity. MRI revealed that allogeneic CDCs attenuated remodeling, improved global and regional function, decreased scar size, and increased viable myocardium compared with placebo 2 months post-treatment. Extensive histological analysis validated quantitatively the MRI measurements of scar size, scar mass, and viable mass. CDCs neither altered gadolinium contrast myocardial kinetics nor induced changes in vascular density or architecture in viable and scarred myocardium. Histology demonstrated that CDCs lead to cardiomyocyte hyperplasia in the border zone, consistent with the observed stimulation of endogenous regenerative mechanisms (cardiomyocyte cycling, upregulation of endogenous progenitors, angiogenesis). Conclusions— Contrast-enhanced MRI accurately measures scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. MRI represents a useful tool for assessing dynamic changes in the infarct and monitoring regenerative efficacy.

Background: Myocardial perfusion scintigraphy (MPS) plays an important role in the evaluation and quantification of myocardial ischemia, and those with significant ischemia (SI) benefit most from revascularization procedures. This study aimed to identify the clinical factors and anatomical features associated with SI in patients with stable ischemic heart disease (SIHD). Methods and Results: Data were analyzed from 4197 SIHD patients undergoing PCI in The Japan Cardiovascular Database (N = 11,258) between September 2008 and April 2013. MPS was used to evaluate 1070 (25.5%) patients. Significant ischemia (SI) was defined as more than 10% ischemic region. Logistic regression analysis was performed to identify any clinical factors associated with SI. Patients with a history of heart failure, stroke, or anginal symptoms with Canadian Cardiovascular Society class 2 or more were more likely to have SI (odds ratio [OR] 1.63, p = 0.025, OR: 1.85, p = 0.009, and OR: 1.49, p = 0.003, respectively). When angiographic variables were considered, a proximal left anterior descending artery (pLAD) lesion was the sole factor associated with SI (OR: 1.46, p = 0.012) (Table). Importantly, those with SI had more complications (p = 0.006), most notably post-PCI infarcts (p = 0.008) (Figure). Conclusions: Patients’ background data, such as stronger anginal symptoms or a pLAD lesion, were associated with SI. Since patients with SI are necessary to be treated with PCI to improve long-term prognosis, however procedure-related complications happen more frequently than non-SI patients. Physicians must give their full attention to the PCI procedure in SI patients to minimize their complication rate.

Background: Myocardial perfusion scintigraphy (MPS) plays a pivotal role in the evaluation of ischemia. Coronary computed tomography angiography (CTA) is emerging to evaluate anatomical features for stable ischemic heart disease (SIHD). This study aimed to identify the clinical characteristics of ischemia (MPS) or anatomy (CTA)-oriented percutaneous coronary intervention (PCI) patients for SIHD. Methods and Results: Data were analyzed from 4197 SIHD patients undergoing PCI in The Japan Cardiovascular Database (N = 11,258) between September 2008 and April 2013. MPS was used to evaluate 1070 (25.5%) patients. Significant ischemia (SI) was defined as more than 10% ischemic region. CTA was performed in 1433 patients, and patients with ischemia evaluation and unavailable results were excluded. A total of 694 patients (16.5%) were evaluated as CTA group. CTA-oriented PCI patients had milder illness in their background (Table). More than half of patients had multivessel disease (CTA vs non-SI vs SI; 51.6, 66.3, 71.9%). Incomplete revascularization ratio was significantly lower in CTA group (0.6, 2.2, 3.1%, p = 0.003). However, CTA-oriented PCI patients and SI patients had significantly more complications than non-SI patients (6.6, 3.8, 8.2%, p = 0.022), especially post-procedural infarction (2.4, 0.5, 3.0%, p = 0.032) (Figure). Conclusions: Even though CTA-oriented PCI patients had incomplete revascularization less frequently, they experienced more complications. These results indicate that functional assessment is important to predict in-hospital complications, and physicians need to give their attention to CTA-oriented PCI patients as well as SI patients.

Introduction: Cardiosphere-derived cells (CDCs) confer both cardioprotection and regeneration in acute myocardial infarction (MI). However, only long-term (>3 wks) post-MI endpoints have been studied, making it impossible to investigate the cardioprotective effects in isolation. Macrophages (MF) concentrate within the heart following ischemic injury as part of the inflammatory response to injury. Here we test the hypothesis that CDCs are cardioprotective by modifying MF within the myocardium post-MI. Methods & Results: Wistar-Kyoto rats (aged 8-12 wks) underwent 45 min of ischemia followed by 20 min of reperfusion, then intracoronary (i.c.) infusion of either saline or CDCs (5x10 5 ). The use of a 48 hr endpoint enabled the selective study of cardioprotection. CDC-treated animals had preserved ejection fraction (59.2% vs. 47.4%; p+ MF (pNos2, Tnf, Il1b ; pIn vitro , MF conditioned by transwell exposure to CDCs (M CDC ) exhibited distinct gene profiles relative to proinflammatory M 1 or “healing” M 2 polarization states (M CDC : Il10 ; M 1 : Nos2 ; M 2 : Arg1, Pparg ). Adoptive transfer of selective MF populations into the heart (i.c.; 20 min post-reflow) revealed that M CDC , but not M 1 or M 2 MF, could recapitulate the reduction in infarct size (M CDC : 4.5%; M 1 : 14.0%; M 2 : 10.8%; pIn vitro co-culture shows that M CDC selectively reduce cardiomyocyte apoptosis following oxidant stress (M CDC : 9.9%; M 1 : 39.4%; M 2 : 37.4%; p Conclusions: CDCs are cardioprotective when administered 20 min after reflow; the timing distinguishes this form of cardioprotection from preconditioning or ischemic postconditioning. Various lines of evidence indicate that CDCs work by polarizing MF toward a cardioprotective phenotype. The findings motivate further translational development of the adjunctive use of CDCs post-MI to limit infarct size.

[Background] Congestive heart failure (CHF) is characterized by activation of the sympathetic nervous system (SNS) with depletion of norepinephrine (NE) stores, which was initially considered to be the result of excess NE secretion and the loss of noradrenergic nerve terminals. Recent studies however have revealed that it is caused by down regulation of NE synthesis and re-uptake, although the molecular mechanism of down regulation of the sympathetic neuronal function remains unknown. We recently found in an animal model of CHF that the cardiac SNS switches the neurotransmitter property from catecholaminergic to cholinergic, mediated by cytokines LIF and CT-1 secreted from failing myocardium. This study was designed to investigate whether or not this cholinergic transdifferentiation of cardiac SNS occurs in patients with CHF. [Methods & Results] (1) We analyzed 8 samples from patients who died of non-cardiac causes obtained at autopsy (control group), and 8 samples from patients with CHF (CHF group). Five of them died of CHF, and 3 were obtained from native hearts of transplant recipients. (2) The heart weight was significantly higher in the CHF group. (3) The gross morphology of the cardiac SNS did not differ between the two groups. HE and Masson trichrome staining showed disorganized cardiomyocytes and interstitial fibrosis in CHF. (4) Immunostaining for tyrosine hydroxylase (TH, sympathetic nerve marker) revealed that the epicardial nerve bundles and stellate ganglia of the control group had a predominance of TH + nerves, whereas those of CHF group were significantly decreased. (5) Immunostaining for choline transporter (CHT, cholinergic neuron marker) revealed that CHT + neurons were markedly increased in the epicardial nerve bundles of CHF hearts compared with the control group. Some nerves co-expressed both TH and CHT markers. (6) Immunostaining for choline acetyl transferase (ChAT, a cholinergic neuron marker) revealed that stellate ganglia had a lot of ChAT + neurons compared with the control. (7) Nissl staining showed that there was no difference between the two groups in neuron number in the stellate ganglia. [Conclusions] These results indicated that in patients with CHF the cardiac sympathetic nerves also had cholinergic nerve properties.

Cardiac nerve sprouting and heterogeneous reinnervation after myocardial infarction (MI) lead to arrhythmogenesis and sudden cardiac death. Despite its importance, little is known about the contribution of the intrinsic cardiac nervous system on reinnervation. We previously reported that neural crest stem cells (NCSC) reside in the heart, and can differentiate into neuron, glia, smooth muscle cells, and cardiomyocytes. The present study scrutinized the developmental origin of intrinsic cardiac adrenergic (ICA) cells, and investigated whether NCSC supply ICA and contribute to reinnervation after MI. (1) We analyzed two distinct double transgenic mice, encoding protein 0-Cre/Floxed-EGFP (P0-EGFP) and dopamine β -hydroxylase-Cre/Floxed-EGFP (DBH-EGFP), to map neural crest-derived cell fate and catecholaminergic cell fate, respectively. ICA cells, demarcated with tyrosine hydroxylase (TH, catecholaminergic marker)-immunopositive cells within ventricles, co-expressed EGFP in both P0-EGFP and DBH-EGFP mice. Most ICA cells were nucleated and round-shaped, although 5% of the cells extended nerve fibers. (2) NCSC were marked with EGFP in P0-EGFP mice, expressed nestin, musashi-1 and p75 nerve growth factor (NGF) receptor, and showed sphere formation. They frequently resided in the outflow tract, but also existed in all parts of the heart. (3) ICA cell number and TH mRNA expression within ventricles were increased in transgenic mice overexpressing NGF in the heart. (4) P0-EGFP mice were subjected to MI by coronary artery occlusion. EGFP-positive cells were markedly increased after MI, and more so in the border zone area than in the non-infarcted area. (5) Similarly, DBH-EGFP subjected to MI revealed an increase in EGFP-positive cells at the border zone area, and some of them extended neurites among the sprouting nerves. (6) TH and EGFP mRNA were increased in DBH-EGFP ventricles, concomitant with cardiac NGF upregulation. These findings demonstrated that ICA cells were derived from neural crest cells, and dependent on NGF synthesized in the heart. NCSC were mobilized to the infarcted myocardium, differentiated into TH-positive nerves, and contributed to reinnervation after MI.

Background: Although prostaglandin I 2 and its derivatives are clinically used for the treatment of pulmonary hypertension, continuous intravenous administration is required for the effective treatment. Prostaglandin I 2 synthase (PGIS) is one of the most powerful therapeutic genes against pulmonary hypertension (PH). The purpose of this study was to investigate whether the human PGIS (hPGIS) gene transfer using adeno-associated virus (AAV) vector into the skeletal muscle was effective to the hypoxia induced-pulmonary hypertension in mice. Methods and Results: PH was induced in the mice subjected to hypoxic chamber (O2 10%) for 8 weeks. Right ventricular systolic pressure (RVSP) significantly increased in PH group compared with control group. In the gene transferred group (AAV1-hPGIS), the AAV type1-hPGIS (2.0×10 11 v.g./body) vector was injected into the left thigh muscle at 24 hour after exposure to hypoxia. Saline injected mice were served as control group. After 8 weeks, hemodynamics and histological analysis was performed. AAV1-PGIS significantly decreased right ventricular systolic pressure to 26±5 mmHg compared with PH group (40±4 mmHg). Systemic blood pressure did not significantly decrease AAV-hPGIS treated group. QT-PCR revealed that PH group increased BNP mRNA in RV by 6-fold higher, while AAV1-hPGIS group decreased to 1.3-fold compared with the control group. Histological analysis revealed that PH group showed severe medial thickening of the pulmonary artery, and increased %wall thickness to 33.3±7.3%, while AAV1-hPGIS decreased to 17±3.6 %. RT-PCR revealed that the hPGIS gene expression was detected only at the injected side, but not in the liver, kidney, heart, and the other organs. Histological analysis showed no pathological changes at the injected skeletal muscles. Finally, Kaplan-Mayer analysis revealed that PH group showed 47% survival at 12 weeks wile AAV1-hPGIS group showed 73%, indicating that AAV1-hPGIS treatment significantly increased the survival of PH. Conclusions: Our study demonstrated that the AAV1-mediaqted gene transfer of PGIS was effective to treat the hypoxia-induced pulmonary hypertension in mice, and can prolong the survival of these animals.

Background : Cardiac hypertrophy induces the fetal isoform of genes (rejuvenation), including contractile proteins, ion channels, and natriuretic peptides. Cardiac sympathetic nerve function is known to be altered in cardiac hypertrophy and congestive heart failure. We recently reported that alteration of cardiac sympathetic nerves (CSN) was caused by their rejuvenation (Circ Res, 2007). The present study was designed to examine the precise characterization of the rejuvenation of CSN in cardiac hypertrophy. Methods and Results : RV hypertrophy was produced by consistent hypoxia (10% O 2 ) in C57/BL6 mice. RV pressure increased to 47 mmHg, and RV/(body weight) ratio increased by 1.6 fold. Nerve growth factor protein was augmented in hypertrophic RV, but was unchanged in LV. Double-transgenic mice, which specifically express eGFP (enhanced green fluorescent protein) in the sympathetic neurons, was generated by crossing dopamine β-hydroxylase (DBH)-Cre mice with Floxed-eGFP mice. The eGFP-positive CSN were markedly increased in hypertrophic RV, but not in LV. Nerve density, quantitated by immunostained area with eGFP and GAP43 (growth-associated corn marker), increased by 8.1 and 9.3 fold, respectively, in RV, but not in LV. (4) Catecholamine content was attenuated in RV. (5) Western blot revealed that tyrosine hydroxylase was markedly down-regulated in RV. (6) Immunostaining clearly demonstrated that the immature neuron markers, PSA-NCAM (highly polysialylated neural cell adhesion molecule) and Ulip-1 (Unc-33-like phosphoprotein 1), were expressed in CSN in hypertrophic RV and stellate ganglia. Basic helix-loop-helix transcription factor, Mash-1 (mammalian achaete-scute complex homolog 1) was strongly expressed in the stellate ganglia. (7) Immature neuron marker-immunopositive cells in stellate ganglia had a markedly decreased TH expression. Conclusion : The rejuvenated CSN showed various immature and fetal neuron marker genes at not only the peripheral axons but also the cellular bodies at the stellate ganglia. Rejuvenation of CSN might be critically involved in the alteration of sympathetic neuronal function in cardiac hypertrophy, including depressed norepinephrine synthesis and hyperinnervation.

Background— Molecular mechanisms regulating the cardiac sensory nervous system remain poorly understood. Cardiac sensory nerve impairment causes silent myocardial ischemia, a main cause of sudden death in diabetes mellitus (DM). The present study focused on the roles of nerve growth factor (NGF) in the regulation of the cardiac sensory nervous system and analyzed the mechanism of silent myocardial ischemia in DM. Methods and Results— We screened neurotrophic factors and found that cardiac sensory nerves developed in parallel with NGF synthesized in the heart. Cardiac nociceptive sensory nerves that were immunopositive for calcitonin gene-related peptide, dorsal root ganglia (DRG), and the dorsal horn were markedly retarded in NGF-deficient mice, whereas cardiac-specific overexpression of NGF rescued these deficits. DM was induced with streptozotocin in wild-type and transgenic mice overexpressing NGF in the heart. Downregulation of NGF, calcitonin gene-related peptide–immunopositive cardiac sensory denervation, and atrophic changes in DRG were observed in DM-induced wild-type mice, whereas these deteriorations were reversed in DM-induced NGF transgenic mice. Cardiac sensory function, measured by myocardial ischemia–induced c-Fos expression in DRG, was also downregulated by DM in the wild-type mice but not in NGF transgenic mice. Direct gene transfer of NGF in the diabetic rat hearts improved impaired cardiac sensory innervation and function, determined by electrophysiological activity of cardiac afferent nerves during myocardial ischemia. Conclusions— These findings demonstrate that the development and regulation of the cardiac sensory nervous system are dependent on NGF synthesized in the heart and that DM-induced NGF reduction causes cardiac sensory neuropathy.