There are limited systematic data on the incidence, clinical characteristics and outcomes of congestive heart failure (CHF) in patients with hyperthyroidism. The aim of this study was to investigate the incidence, clinical characteristics and outcome of CHF as the initial presentation in patients with primary hyperthyroidism.
The prevalence, clinical characteristics and outcome of CHF was studied in 591 consecutive patients (mean (SD) age 45 (1) years, 140 men) who presented with primary hyperthyroidism.
CHF was the presenting condition in 34 patients (5.8%) with hyperthyroidism. The presence of atrial fibrillation at presentation (OR 37.4, 95% CI 9.72 to 144.0, p<0.001) was an independent predictor for the occurrence of CHF. Of the 34 patients with CHF, 16 (47%) had systolic left ventricular dysfunction with left ventricular ejection fraction (LVEF)<50%. They were predominantly male (OR 26.6, 95% CI 2.6 to 272.5, p = 0.006) and had a lower serum thyroxine level (OR 0.93, 95% CI 0.87 to 0.99, p = 0.044) than patients with preserved left ventricular systolic function. In these patients, LVEF (55 (4)% vs 30 (2)%, p<0.001) and New York Heart Association functional class (1.2 (0.1) vs 2.5 (0.2), p<0.001) improved significantly 3 months after achieving euthyroid status. Systolic left ventricular dysfunction (mean (SD) LVEF 38 (4)%) persisted on long‐term follow‐up in five patients: no clinical parameter could be identified to predict the occurrence of this persistent cardiomyopathy (p>0.05).
CHF was the initial clinical presentation in approximately 6% of patients with hyperthyroidism, and half of them had left ventricular systolic dysfunction. Symptoms of CHF subsided and LVEF improved after treatment for hyperthyroidism. Nonetheless, one‐third of these patients developed persistent dilated cardiomyopathy.
Type 2 diabetes mellitus (T2DM) is associated with subclinical myocardial injury although the underlying mechanism is uncertain. We postulated that arterial stiffness, endothelial dysfunction and subclinical atherosclerosis may contribute to subclinical myocardial injury in patients with T2DM.
Serum high-sensitivity troponin I (hs-TNI) an indicator of myocardial injury, was measured in 100 patients with T2DM without clinical evidence of macrovascular disease and 150 age and gender-matched controls. Elevated hs-TnI was defined as follow (derived from the 99th percentile from controls): Male >11.1 ng/L; female >7.6 ng/L. Measures that may contribute to myocardial damage in patients with T2DM, including brachial-ankle pulse wave velocity (ba-PWV), brachial flow mediated dilatation (FMD) and carotid intima media thickness (IMT), were also assessed.
The serum level of hs-TNI (5.7±9.2 μg/L vs. 3.2±1.9 μg/L, P< 0.01) and the prevalence of elevated hs-TNI (12% vs. 4%, P = 0.02) were significantly higher in patients with T2DM than controls. Patients with T2DM also had significantly worse ba-PWV (17.98±3.91ms-1 vs. 15.70±2.96 ms-1), brachial FMD (2.6±3.5% vs. 5.5±4.2%, P< 0.01) and carotid IMT (0.96±0.20 mm vs. 0.86±0.14 mm, P< 0.01). In patients with T2DM, hs-TNI was positively correlated with systolic blood pressure (r = 0.31, P<0.01), serum creatinine (r = 0.26, P = 0.01) and ba-PWV (r = 0.34, P< 0.01). Importantly, multiple regression revealed that only ba-PWV was independently associated with hs-TNI (β = 0.25, P = 0.04).
The results demonstrated an independent association between ba-PWV and hs-TNI in patients with T2DM with no clinical evidence of macrovascular disease. These findings suggest that increased arterial stiffness is closely related to subclinical myocardial injury in patients with T2DM.
Type 2 diabetes mellitus; Myocardial injury; Arterial stiffiness; High-sensitivity troponin I
Endothelial progenitor cells (EPCs) are responsible for angiogenesis and maintenance of microvascular integrity, the number of EPCs is correlated with oxidative stress. Their relation to myocardial dysfunction in patients with type 2 diabetes mellitus (T2DM) is nonetheless unknown.
Eighty-seven patients with T2DM and no history of coronary artery disease were recruited. Transthoracic echocardiography and detailed evaluation of left ventricular (LV) systolic function by 2-dimensional (2D) speckle tracking derived strain analysis in 3 orthogonal directions was performed. Four subpopulations of EPCs, including CD34+, CD133+, CD34+/kinase insert domain-containing receptor (KDR) + and CD133+/KDR + EPCs, were measured by flow cytometry. Oxidative stress was assessed by superoxide dismutase (SOD).
The mean age of the patients was 62 ± 9 years and 39.6% were male. Those with an impaired longitudinal strain had a lower number of CD34+ EPCs (2.82 ± 1.87% vs. 3.74 ± 2.12%, P < 0.05) than those with preserved longitudinal strain. When compared with those with preserved circumferential strain, patients with an impaired circumferential strain had a lower number of CD34+ EPCs (2.63 ± 1.80% vs. 3.87 ± 2.10%, P < 0.01) and SOD level (0.13 ± 0.06U/ml vs. 0.20 ± 0.08U/ml, P < 0.01). Patients with an impaired radial strain nonetheless had a lower number of CD34+ EPCs (2.62 ± 2.08% vs. 3.69 ± 1.99%, P < 0.05). Multivariate analysis demonstrated that only impaired global circumferential strain remained significantly associated with CD34 + EPCs and SOD.
LV global circumferential strain was independently associated with number of CD34+ EPCs and SOD. These findings suggest that myocardial dysfunction in patients with T2DM is related to depletion of EPCs and increased oxidative stress.
Type 2 diabetes mellitus; Myocardial injury; Endothelial progenitor cells
We identified an autosomal dominant non-sense mutation (R225X) in exon 4 of the lamin A/C (LMNA) gene in a Chinese family spanning 3 generations with familial dilated cardiomyopathy (DCM). In present study, we aim to generate induced pluripotent stem cells derived cardiomyocytes (iPSC-CMs) from an affected patient with R225X and another patient bearing LMNA frame-shift mutation for drug screening.
METHODS and RESULTS
Higher prevalence of nuclear bleb formation and micronucleation was present in LMNAR225X/WT and LMNAFramshift/WT iPSC-CMs. Under field electrical stimulation, percentage of LMNA-mutated iPSC-CMs exhibiting nuclear senescence and cellular apoptosis markedly increased. shRNA knockdown of LMNA replicated those phenotypes of the mutated LMNA field electrical stress. Pharmacological blockade of ERK1/2 pathway with MEK1/2 inhibitors, U0126 and selumetinib (AZD6244) significantly attenuated the pro-apoptotic effects of field electric stimulation on the mutated LMNA iPSC-CMs.
LMNA-related DCM was modeled in-vitro using patient-specific iPSC-CMs. Our results demonstrated that haploinsufficiency due to R225X LMNA non-sense mutation was associated with accelerated nuclear senescence and apoptosis of iPSC- CMs under electrical stimulation, which can be significantly attenuated by therapeutic blockade of stress-related ERK1/2 pathway.
Dilated cardiomyopathy; induced pluripotent stem cells; LMNA
Previous experimental studies suggested that mesenchymal stem cell transplantation causes cardiac nerve sprouting; however, whether bone marrow (BM)-derived mononuclear cells (MNC) and endothelial progenitor cells (EPC) can also lead to cardiac nerve sprouting and alter gap junction expression remains unclear. We investigated the effect of electroanatomical mapping-guided direct intramyocardial transplantation of BM-MNC (n = 8) and CD31+EPC (n = 8) compared with saline control (n = 8) on cardiac nerve sprouting and gap junction expression in a swine model of chronic ischemic myocardium. At 12 weeks after transplantation, the distribution and density of cardiac nerve sprouting were determined by staining of tyrosine hydroxylase (TH) and growth associated protein 43(GAP-43) and expression of connexin 43 in the targeted ischemic and remote normal myocardium. After 12 weeks, no animal developed sudden death after the transplantation. There were no significant differences in the number of cells with positive staining of TH and GAP-43 in the ischemic and normal myocardium between three groups. Furthermore, expression of connexin 43 was also similar in the ischemic and normal myocardia in each group of animals (P > 0.05). The results of this study demonstrated that intramyocardial BM-derived MNC or EPC transplantation in a large animal model of chronic myocardial ischemia was not associated with increased cardiac nerve sprouting over the ischemic myocardium.
Bone marrow cells; Ischemia; Arrhythmia; Nerve sprouting; Connecxin 43
Patients with type 2 diabetes mellitus (DM) have increased risk of endothelial dysfunction and arterial stiffness. Levels of circulating endothelial progenitor cells (EPCs) are also reduced in hyperglycemic states. However, the relationships between glycemic control, levels of EPCs and arterial stiffness are unknown.
We measured circulating EPCs and brachial-ankle pulse wave velocity (baPWV) in 234 patients with type 2 DM and compared them with 121 age- and sex-matched controls.
Patients with DM had significantly lower circulating Log CD34/KDR+ and Log CD133/KDR+ EPC counts, and higher Log baPWV compared with controls (all P < 0.05). Among those 120/234 (51%) of DM patients with satisfactory glycemic control (defined by Hemoglobin A1c, HbA1c < 6.5%), they had significantly higher circulating Log CD34/KDR+ and Log CD133/KDR+ EPC counts, and lower Log baPWV compared with patients with poor glycemic control (all P < 0.05). The circulating levels of Log CD34/KDR+ EPC (r = -0.46, P < 0.001) and Log CD133/KDR+ EPC counts (r = -0.45, P < 0.001) were negatively correlated with Log baPWV. Whilst the level of HbA1c positively correlated with Log baPWV (r = 0.20, P < 0.05) and negatively correlated with circulating levels of Log CD34/KDR+ EPC (r = -0.40, P < 0.001) and Log CD133/KDR+ EPC (r = -0.41, P < 0.001). Multivariate analysis revealed that HbA1c, Log CD34/KDR+ and Log CD133/KDR+ EPC counts were independent predictors of Log baPWV (P < 0.05).
In patients with type 2 DM, the level of circulating EPCs and arterial stiffness were closely related to their glycemic control. Furthermore, DM patients with satisfactory glycemic control had higher levels of circulating EPCs and were associated with lower arterial stiffness.
A better understanding of the ionic mechanisms for cardiac automaticity can lead to better strategies for engineering bio-artificial pacemakers. Here, we attempted to better define the relative contribution of If and IK1 in the generation of spontaneous action potentials (SAPs) in cardiomyocytes (CMs).
Methods and results
Monolayers of neonatal rat ventricular myocytes (NRVMs) were transduced with a recombinant adenovirus (Ad) to express a gating-engineered HCN1 construct (HCN1-ΔΔΔ) for patch-clamp and multielectrode array (MEA) recordings. Single NRVMs exhibited a bi-phasic response in the generation of SAPs (62.6 ± 17.4 b.p.m., Days 1–2; 194.3 ± 12.3 b.p.m., Days 3–4; 73% quiescent, Days 9–10). Although automaticity time-dependently decreased and subsequently ceased, If remained fairly stable (−5.2 ± 1.1 pA/pF, Days 1–2; −5.1 ± 1.4 pA/pF, Days 7–8; −4.3 ± 1.3 pA/pF, Days 13–14). In contrast, IK1 declined rapidly (from −16.9 ± 2.7 pA/pF on Days 1–2 to −4.4 ± 1.6 pA/pF on Days 5–6). Maximum diastolic potential/resting membrane potential (r = 0.89) and action potential duration at 50% (APD50, r = 0.73) and 90% (APD90, r = 0.75) but not the firing rate (r = −0.3) were positively correlated to the IK1. Similarly, monolayer NRVMs ceased to spontaneously fire after long-term culture. Ad-HCN1-ΔΔΔ transduction restored pacing in silenced individual and monolayer NRVMs but with reduced conduction velocity and field potential amplitude.
We conclude that the combination of IK1 and If primes CMs for bio-artificial pacing by determining the threshold. However, If functions as a membrane potential oscillator to determine the basal firing frequency. Future engineering of automaticity in the multicellular setting needs to have conduction taken into consideration.
If; IK1; HCN; Automaticity; Action potential
Cardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis, the key process underlying excitation-contraction coupling.
We investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs).
Methods and Results
We differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes, spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore, in contrast with hESC-derived cardiomyocytes, ryanodine did not reduce the amplitudes, maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition, spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2), sacroplasmic recticulum calcium-ATPase (SERCA), junction (Jun) and triadin (TRDN), were significantly lower in hiPSC than in hESCs.
The results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts.
Electronic supplementary material
The online version of this article (doi:10.1007/s12015-011-9273-3) contains supplementary material, which is available to authorized users.
Human induced pluripotent stem cells; Cardiomyocytes; Calcium handling
The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope, mostly the lamins. Lamins are the main constituents of the nuclear lamina, a filamentous meshwork associated with the inner nuclear membrane that provides mechanical stability and plays important roles in processes such as transcription, DNA replication and chromatin organization. More than 300 mutations in lamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient with inherited dilated cardiomiopathy and 2 patients with distinct accelerated forms of aging, atypical Werner syndrome and Hutchinson Gilford progeria, all of which are caused by mutations in lamin A/C. These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts. Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases.
reprogramming; induced pluripotent stem cells; lamin A/C; dilated cardiomyopathy; atypical Werner syndrome; Hutchinson Gilford progeria
Human-induced pluripotent stem cells (iPSCs) generated from human adult somatic cells through reprogramming hold great promises for future regenerative medicine. However, exposure of human iPSCs to animal feeder and serum in the process of their generation and maintenance imposes risk of transmitting animal pathogens to human subjects, thus hindering the potential therapeutic applications. Here, we report the successful generation of human iPSCs in a feeder-independent culture system with defined factors. Two stable human iPSC lines were established from primary human dermal fibroblasts of two healthy volunteers. These human iPSCs expressed a panel of pluripotency markers including stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-60, TRA-1-81, and alkaline phosphatase, while maintaining normal karyotypes and the exogenous reprogramming factors being silenced. In addition, these human iPSCs can differentiate along lineages representative of the three embryonic germ layers upon formation of embryoid bodies, indicating their pluripotency. Furthermore, subcutaneous transplantation of these cells into immunodeficient mice resulted in teratoma formation in 6 to 8 weeks. Our findings are an important step toward generating patient-specific iPSCs in a more clinically compliant manner by eliminating the need of animal feeder cells and animal serum.
Normal heart rhythms originate in the sinoatrial node. HCN-encoded funny current (If) and the Kir2-encoded inward rectifier (IK1) counteract each other by respectively oscillating and stabilizing the negative resting membrane potential, controlling action potential firing. Therefore, IK1 suppression and If overexpression have been independently exploited to convert cardiomyocytes (CMs) into AP-firing bioartificial pacemakers. Although the two strategies have been largely assumed synergistic, their complementarity has not been investigated.
Methods and Results
We explored the inter-relationships of automaticity, If and IK1 by transducing single left ventricular (LV) CMs isolated from guinea pig hearts with the recombinant adenoviruses Ad-CMV-GFP-IRES-HCN1-ΔΔΔ and/or Ad-CGI-Kir2.1 to mediate their current densities via whole-cell patch clamp technique at 37°C. Results showed that Ad-CGI-HCN1-ΔΔΔ- but not Ad-CGI-Kir2.1-transduction induced automaticity (181.1±13.1 bpm). Interestingly, Ad-CGI-HCN1-ΔΔΔ/Ad-CGI-Kir2.1 cotransduction significantly promoted the induced firing frequency (320.0±15.8 bpm; p<0.05). Correlation analysis revealed that the firing frequency, phase 4 slope and APD90 of AP-firing LV CMs were correlated to If (R2>0.7) only when -2>IK1>-4 pA/pF but not to IK1 over the entire If ranges examined (0.02If>-4 pA/pF. As anticipated, however, APD90 was correlated to IK1 (R2=0.4).
We conclude that an optimal level of IK1 maintains a voltage range for If to operate most effectively during a dynamic cardiac cycle.
If; IK1; bioartificial pacemaker; automaticity; synergism
Over the last five decades, pacemaker therapy has undergone remarkable technological advances with increasing sophistication of pacemaker features. However, device longevity has remained one of the major issues in pacemaker design ever since the first endocardial pacing lead implantation in 1958. In addition to various hardware design to enhance device longevity, software-based solutions to minimize pacing energy and yet with good safety margin have also been developed. Together with desire and need of fully automatic pacing system in increasingly busy pacemaker clinic, several manufacturers have introduced different automatic threshold management algorithm. This article summarizes the current state-of-the-art management in pacing threshold in the modern pacemakers.
Pacemaker; Threshold management; Autocapture
The hyperpolarization-activated cyclic nucleotide-modulated channel gene family (HCN1-4) encodes the membrane depolarizing current that underlies pacemaking. Although the topology of HCN resembles Kv channels, much less is known about their structure-function correlation. Previously, we identified several pore residues in the S5-P linker and P-loop that are externally accessible and/or influence HCN gating, and proposed an evolutionarily conserved pore-to-gate mechanism. Here we sought dynamic evidence by assessing the functional consequences of Cys-scanning substitutions in the unexplored P-S6 linker (residues 352–359), the HCN1-R background (that is, resistant to sulfhydryl-reactive agents). None of A352C, Q353C, A354C, P355C, V356C, S357C, M358C, or S359C produced functional currents; the loss-of-function of Q353C, A354C, S357C, and M358C could be rescued by the reducing agent dithiothreitol. Q353C, A354C, and S357C, but not M358C and HCN1-R, were sensitive to Cd2+ blockade (IC50 = 3–12 μM vs. >1 mM). External application of the positively charged covalent sulfhydryl modifier MTSET irreversibly reduced I−140mV of Q353C and A354C to 27.9 ± 3.4% and 58.2 ± 13.1% of the control, respectively, and caused significant steady-state activation shifts (∆V1/2 = –21.1 ± 1.6 for Q353C and −10.0 ± 2.9 mV for A354C). Interestingly, MTSET reactivity was also state dependent. MTSET, however, affected neither S357C nor M358C, indicating site specificity. Collectively, we have identified novel P-S6 residues whose extracellular accessibility was sterically and state dependent and have provided the first functional evidence consistent with a dynamic HCN pore-to-gate model.
Outer pore; Pacemaker channels; HCN
Although If, encoded by the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channel gene family, is known to be functionally important in pacing, its mechanistic action is largely inferential and indeed somewhat controversial. To dissect in detail the role of If, we investigated the functional consequences of overexpressing in adult guinea pig left ventricular cardiomyocytes (LVCMs) various HCN1 constructs that have been engineered to exhibit different gating properties.
Methods and Results
We created the recombinant adenoviruses Ad-CMV-GFP-IRES (CGI), Ad-CGI-HCN1, Ad-CGI-HCN1-ΔΔΔ, and Ad-CGI-HCN1-Ins, which mediate ectopic expression of GFP alone, WT, EVY235-7ΔΔΔ, and Ins HCN1 channels, respectively; EVY235-7ΔΔΔ and Ins encode channels in which the S3—S4 linkers have been shortened and lengthened to favor and inhibit opening, respectively. Ad-CGI-HCN1, Ad-CGI-HCN1-ΔΔΔ, and Ad-CGI-HCN1-Ins, but not control Ad-CGI, transduction of LVCMs led to robust expression of If with comparable densities when fully open (≈-22 pA/pF at -140 mV; P>0.05) but distinctive activation profiles (V1/2=-70.8±0.6, -60.4±0.7, and -87.7±0.7 mV; P<0.01, respectively). Whereas control (nontransduced or Ad-CGI—transduced) LVCMs were electrically quiescent, automaticity (206±16 bpm) was observed exclusively in 61% of Ad-HCN1-ΔΔΔ—transduced cells that displayed depolarized maximum diastolic potential (-60.6±0.5 versus -70.6±0.6 mV of resting membrane potential of control cells; P<0.01) and gradual phase 4 depolarization (306±32 mV/s) that were typical of genuine nodal cells. Furthermore, spontaneously firing Ad-HCN1-ΔΔΔ—transduced LVCMs responded positively to adrenergic stimulation (P<0.05) but exhibited neither overdrive excitation nor suppression. In contrast, the remaining 39% of Ad-HCN1-ΔΔΔ—transduced cells exhibited no spontaneous action potentials; however, a single ventricular action potential associated with a depolarized resting membrane potential and a unique, incomplete “phase 4—like” depolarization that did not lead to subsequent firing could be elicited on simulation. Such an intermediate phenotype, similarly observed in 100% of Ad-CGI-HCN— and Ad-CGI-HCN1-Ins—transduced LVCMs, could be readily reversed by ZD7288, hinting at a direct role of If. Correlation analysis revealed the specific biophysical parameters required for If to function as an active membrane potential oscillator.
Our results not only contribute to a better understanding of cardiac pacing but also may advance current efforts that focus primarily on automaticity induction to the next level by enabling bioengineering of central and peripheral cells that make up the native sinoatrial node.
genes; ion channels; pacemakers; sinoatrial node; tissue engineering
Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC), human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC), and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless, BM-EC, hESC-EC and hiPSC-EC exhibited typical cobblestone morphology, had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein, and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (P>0.05). While increased expression of major angiogenic factors including epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor, placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (P<0.05), the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (P<0.05). Compared with medium, transplanting BM-EC (n = 6), HUVEC (n = 6), hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion, functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as “off-the-shelf” format for the treatment of tissue ischemia.
Type 2 diabetes mellitus (T2DM) patients are at increased risk of developing cardiovascular events. Unfortunately traditional risk assessment scores, including the Framingham Risk Score (FRS), have only modest accuracy in cardiovascular risk prediction in these patients.
We sought to determine the prognostic values of different non-invasive markers of atherosclerosis, including brachial artery endothelial function, carotid artery atheroma burden, ankle-brachial index, arterial stiffness and computed tomography coronary artery calcium score (CACS) in 151 T2DM Chinese patients that were identified low-intermediate risk from the FRS recalibrated for Chinese (<20% risk in 10 years). Patients were prospectively followed-up and presence of atherosclerotic events documented for a mean duration of 61 ± 16 months.
A total of 17 atherosclerotic events in 16 patients (11%) occurred during the follow-up period. The mean FRS of the study population was 5.0 ± 4.6% and area under curve (AUC) from receiver operating characteristic curve analysis for prediction of atherosclerotic events was 0.59 ± 0.07 (P = 0.21). Among different vascular assessments, CACS > 40 had the best prognostic value (AUC 0.81 ± 0.06, P < 0.01) and offered significantly better accuracy in prediction compared with FRS (P = 0.038 for AUC comparisons). Combination of FRS with CACS or other surrogate vascular markers did not further improve the prognostic values over CACS alone. Multivariate Cox regression analysis identified CACS > 40 as an independent predictor of atherosclerotic events in T2DM patients (Hazards Ratio 27.11, 95% Confidence Interval 3.36-218.81, P = 0.002).
In T2DM patients identified as low-intermediate risk by the FRS, a raised CACS > 40 was an independent predictor for atherosclerotic events.
Vascular markers of atherosclerosis; Type 2 diabetes mellitus
The cardioprotective effects of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A1 (apoA-I) are well documented, but their effects in the direction of the cardiac differentiation of embryonic stem cells are unknown. We evaluated the effects of exogenous apoA-I expression on cardiac differentiation of ESCs and maturation of ESC-derived cardiomyocytes. We stably over-expressed full-length human apoA-I cDNA with lentivirus (LV)-mediated gene transfer in undifferentiated mouse ESCs and human induced pluripotent stem cells. Upon cardiac differentiation, we observed a significantly higher percentage of beating embryoid bodies, an increased number of cardiomyocytes as determined by flow cytometry, and expression of cardiac markers including α-myosin heavy chain, β-myosin heavy chain and myosin light chain 2 ventricular transcripts in LV-apoA-I transduced ESCs compared with control (LV-GFP). In the presence of noggin, a BMP4 antagonist, activation of BMP4-SMAD signaling cascade in apoA-I transduced ESCs completely abolished the apoA-I stimulated cardiac differentiation. Furthermore, co-application of recombinant apoA-I and BMP4 synergistically increased the percentage of beating EBs derived from untransduced D3 ESCs. These together suggests that that pro-cardiogenic apoA-I is mediated via the BMP4-SMAD signaling pathway. Functionally, cardiomyocytes derived from the apoA-I-transduced cells exhibited improved calcium handling properties in both non-caffeine and caffeine-induced calcium transient, suggesting that apoA-I plays a role in enhancing cardiac maturation. This increased cardiac differentiation and maturation has also been observed in human iPSCs, providing further evidence of the beneficial effects of apoA-I in promoting cardiac differentiation. In Conclusion, we present novel experimental evidence that apoA-I enhances cardiac differentiation of ESCs and iPSCs and promotes maturation of the calcium handling property of ESC-derived cardiomyocytes via the BMP4/SMAD signaling pathway.
Mouse (m) and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are known to exhibit immature Ca2+ dynamics such as small whole-cell peak amplitude and slower kinetics relative to those of adult. In this study, we examined the maturity and efficiency of Ca2+-induced Ca2+ release in m and hESC-CMs, the presence of transverse (t) tubules and its effects on the regional Ca2+ dynamics. In m and hESC-CMs, fluorescent staining and atomic force microscopy (AFM) were used to detect the presence of t-tubules, caveolin-3, amphiphysin-2 and colocalization of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs). To avoid ambiguities, regional electrically-stimulated Ca2+ dynamics of single ESC-CMs, rather than spontaneously beating clusters, were measured using confocal microscopy. m and hESC-CMs showed absence of dyads, with neither t-tubules nor colocalization of DHPRs and RyRs. Caveolin-3 and amphiphysin-2, crucial for the biogenesis of t-tubules with robust expression in adult CMs, were also absent. Single m and hESC-CMs displayed non-uniform Ca2+ dynamics across the cell that is typical of CMs deficient of t-tubules. Local Ca2+ transients exhibited greater peak amplitude at the peripheral than at the central region for m (3.50 ± 0.42 vs. 3.05 ± 0.38) and hESC-CMs (2.96 ± 0.25 vs. 2.72 ± 0.25). Kinetically, both the rates of rise to peak amplitude and transient decay were faster for the peripheral relative to the central region. Immature m and hESC-CMs display unsynchronized Ca2+ transients due to the absence of t-tubules and gene products crucial for their biogenesis. Our results provide insights for driving the maturation of ESC-CMs.