Arjang Ruhparwar

Regulation of the Human Cardiac Mitochondrial Ca2+ Uptake by 2 Different Voltage-Gated Ca2+ Channels

Background— Impairment of intracellular Ca2+ homeostasis and mitochondrial function has been implicated in the development of cardiomyopathy. Mitochondrial Ca2+ uptake is thought to be mediated by the Ca2+ uniporter (MCU) and a thus far speculative non-MCU pathway. However, the identity and properties of these pathways are a matter of intense debate, and possible functional alterations in diseased states have remained elusive. Methods and Results— By patch clamping the inner membrane of mitochondria from nonfailing and failing human hearts, we have identified 2 previously unknown Ca2+-selective channels, referred to as mCa1 and mCa2. Both channels are voltage dependent but differ significantly in gating parameters. Compared with mCa2 channels, mCa1 channels exhibit a higher single-channel amplitude, shorter openings, a lower open probability, and 3 to 5 subconductance states. Similar to the MCU, mCa1 is inhibited by 200 nmol/L ruthenium 360, whereas mCa2 is insensitive to 200 nmol/L ruthenium 360 and reduced only by very high concentrations (10 &mgr;mol/L). Both mitochondrial Ca2+ channels are unaffected by blockers of other possibly Ca2+-conducting mitochondrial pores but were activated by spermine (1 mmol/L). Notably, activity of mCa1 and mCa2 channels is decreased in failing compared with nonfailing heart conditions, making them less effective for Ca2+ uptake and likely Ca2+-induced metabolism. Conclusions— Thus, we conclude that the human mitochondrial Ca2+ uptake is mediated by these 2 distinct Ca2+ channels, which are functionally impaired in heart failure. Current properties reveal that the mCa1 channel underlies the human MCU and that the mCa2 channel is responsible for the ruthenium red–insensitive/low-sensitivity non-MCU–type mitochondrial Ca2+ uptake.

Transplanted fetal cardiomyocytes as cardiac pacemaker

BACKGROUND While morphologic integration of transplanted fetal cardiomyocytes into the ventricular myocardium is a well-known fact, no studies have yet shown transplanted cells to coherently contribute to contraction and electrical excitation of the host myocardium. The aim of this study was to prove the hypothesis that by transplanting cardiomyocytes with a higher intrinsic rhythmic rate into the myocardium of the left ventricle, these cells could act as an ectopic pacemaker by functional coupling with host cardiomyocytes. METHODS AND RESULTS Dissociated fetal canine atrial cardiomyocytes including sinus nodal cells were delivered into the free wall of the left ventricle of adult canine X-linked muscular dystrophy dogs (n=2). These dogs fail to express Dystrophin in both cardiac and skeletal muscle. In the control group (n=2) fetal skin fibroblasts were used for grafting. A total of 3-4 weeks after transplantation the dogs underwent catheter ablation of the atrioventricular node (AV-node) and subsequent electrophysiological mapping studies. Transplanted cells were identified by Dystrophin immunoreactivity, indicating survival and morphological integration in the recipient heart. The expression of Connexin 43 between donor and recipient cells suggested formation of gap junctions between injected and host cardiomyocytes. After catheter ablation of the AV-node, a ventricular escape rhythm emerged driving the pace of the heart and originating from the labeled transplantation site. This effect could not be observed in the control group (n=2). CONCLUSIONS The results constitute the first observation of phenomena indicating electrical and mechanical coupling between allogeneic donor cardiomyocytes and recipient myocardium in-vivo. Further experiments are necessary to evaluate the technique as a potential therapy for atrioventricular block.

Upregulation of K(2P)3.1 K+ Current Causes Action Potential Shortening in Patients With Chronic Atrial Fibrillation

Background— Antiarrhythmic management of atrial fibrillation (AF) remains a major clinical challenge. Mechanism-based approaches to AF therapy are sought to increase effectiveness and to provide individualized patient care. K2P3.1 (TASK-1 [tandem of P domains in a weak inward-rectifying K+ channel–related acid-sensitive K+ channel-1]) 2-pore-domain K+ (K2P) channels have been implicated in action potential regulation in animal models. However, their role in the pathophysiology and treatment of paroxysmal and chronic patients with AF is unknown. Methods and Results— Right and left atrial tissue was obtained from patients with paroxysmal or chronic AF and from control subjects in sinus rhythm. Ion channel expression was analyzed by quantitative real-time polymerase chain reaction and Western blot. Membrane currents and action potentials were recorded using voltage- and current-clamp techniques. K2P3.1 subunits exhibited predominantly atrial expression, and atrial K2P3.1 transcript levels were highest among functional K2P channels. K2P3.1 mRNA and protein levels were increased in chronic AF. Enhancement of corresponding currents in the right atrium resulted in shortened action potential duration at 90% of repolarization (APD90) compared with patients in sinus rhythm. In contrast, K2P3.1 expression was not significantly affected in subjects with paroxysmal AF. Pharmacological K2P3.1 inhibition prolonged APD90 in atrial myocytes from patients with chronic AF to values observed among control subjects in sinus rhythm. Conclusions— Enhancement of atrium-selective K2P3.1 currents contributes to APD shortening in patients with chronic AF, and K2P3.1 channel inhibition reverses AF-related APD shortening. These results highlight the potential of K2P3.1 as a novel drug target for mechanism-based AF therapy.

Off-bypass coronary bypass grafting via minithoracotomy using mechanical epicardial stabilization.

BACKGROUND Minimally or less invasive surgical coronary revascularization has gained increasing interest along with new techniques and devices designed for easier and safer procedures. Until recently, it appeared questionable whether grafting techniques with avoidance of cardiopulmonary bypass techniques would allow adequate results compared with conventional techniques using cardioplegic arrest. METHODS Since June 1996, minimally invasive direct coronary artery bypass grafting procedures without cardiopulmonary bypass were intended in 24 patients (19 male, 5 female; age, 60.5 +/- 10.5 years) applying a special system (CardioThoracic Systems, Inc) for internal mammary artery access and epicardial surface stabilization approaching through an anterolateral minithoracotomy. Neither video-assisted preparation nor additional pharmacologic stabilization was applied. Concomitant risk factors and associated comorbidity were frequent. RESULTS The procedure was completed in 23 patients, grafting the left anterior descending coronary artery (n = 21) or diagonal branches (n = 3, 1 sequential) as scheduled. In 1 case with internal mammary artery dissection, cardiopulmonary bypass and sternotomy became necessary. Simultaneous carotid endarterectomy was performed in 1 patient. There were two episodes of intraoperative ventricular fibrillation; no other major complications occurred. Postoperative evaluation was obtained in 16 patients (15 by angiography, 1 by Doppler echocardiography) so far and revealed adequate graft function and patency. CONCLUSIONS Using specially designed instruments for internal mammary artery access and epicardial surface stabilization, minimally invasive direct coronary artery bypass grafting procedures via a minithoracotomy avoiding cardiopulmonary bypass techniques may be applied safely and successfully, even in increased risk constellations.

S100A1 Genetically Targeted Therapy Reverses Dysfunction of Human Failing Cardiomyocytes

OBJECTIVES This study investigated the hypothesis whether S100A1 gene therapy can improve pathological key features in human failing ventricular cardiomyocytes (HFCMs). BACKGROUND Depletion of the Ca²⁺-sensor protein S100A1 drives deterioration of cardiac performance toward heart failure (HF) in experimental animal models. Targeted repair of this molecular defect by cardiac-specific S100A1 gene therapy rescued cardiac performance, raising the immanent question of its effects in human failing myocardium. METHODS Enzymatically isolated HFCMs from hearts with severe systolic HF were subjected to S100A1 and control adenoviral gene transfer and contractile performance, calcium handling, signaling, and energy homeostasis were analyzed by video-edge-detection, FURA2-based epifluorescent microscopy, phosphorylation site-specific antibodies, and mitochondrial assays, respectively. RESULTS Genetically targeted therapy employing the human S100A1 cDNA normalized decreased S100A1 protein levels in HFCMs, reversed both contractile dysfunction and negative force-frequency relationship, and improved contractile reserve under beta-adrenergic receptor (β-AR) stimulation independent of cAMP-dependent (PKA) and calmodulin-dependent (CaMKII) kinase activity. S100A1 reversed underlying Ca²⁺ handling abnormalities basally and under β-AR stimulation shown by improved SR Ca²⁺ handling, intracellular Ca²⁺ transients, diastolic Ca²⁺ overload, and diminished susceptibility to arrhythmogenic SR Ca²⁺ leak, respectively. Moreover, S100A1 ameliorated compromised mitochondrial function and restored the phosphocreatine/adenosine-triphosphate ratio. CONCLUSIONS Our results demonstrate for the first time the therapeutic efficacy of genetically reconstituted S100A1 protein levels in HFCMs by reversing pathophysiological features that characterize human failing myocardium. Our findings close a gap in our understanding of S100A1s effects in human cardiomyocytes and strengthen the rationale for future molecular-guided therapy of human HF.

Transplanted human cord blood-derived unrestricted somatic stem cells improve left-ventricular function and prevent left-ventricular dilation and scar formation after acute myocardial infarction

Objective: Functional improvement after acute myocardial ischaemia (MI) has been achieved by transplantation of different adult stem and progenitor cell types. It is controversial whether these cell types are able to form novel functional myocardium. Alternatively, graft-related or immune-related paracrine mechanisms may preserve existing myocardium, improve neovascularisation, affect tissue remodelling or induce endogenous de novo formation of functional myocardium. We have applied an alternative somatic cell type, human cord-blood-derived unrestricted somatic stem cells (USSCs) in a porcine model of acute MI. Methods: USSCs were transplanted into the acutely ischaemic lateral wall of the left ventricle (LV). LV dimension and function were assessed by transoesophageal echocardiography (TEE) pre-MI, immediately post-MI, 48 hours and 8 weeks after USSC injection. Additionally, apoptosis, mitosis and recruitment of macrophages were examined 48 hours post-engraftment. Results: Gender-specific and species-specific FISH/immunostaining failed to detect engrafted donor cells 8 weeks post-MI. Nevertheless, cell treatment effectively preserved natural myocardial architecture. Global left ventricular ejection fraction (LVEF) before MI was 60% (7%). Post-MI, LVEF decreased to 34% (8%). After 8 weeks, LVEF had further decreased to 27% (6%) in the control group and recovered to 52% (2%) in the USSC group (p<0.01). Left-ventricular end-diastolic volume (LVEDV) before MI was 28 (2) ml. 8 weeks post-MI, LVEDV had increased to 77 (4) ml in the control group. No LV dilation was detected in the USSC group (LVEDV: 26 (2) ml, p<0.01). Neither apoptosis nor recruitment of macrophages and mitosis were different in either groups. Conclusions: Transplantation of USSCs significantly improved LV function and prevented scar formation as well as LV dilation. Since differentiation, apoptosis and macrophage mobilisation at infarct site were excluded as underlying mechanisms, paracrine effects are most likely to account for the observed effects of USSC treatment.

Anticoagulation for prosthetic heart valves during pregnancy: is low-molecular-weight heparin an alternative?

We report on the treatment failure of low molecular weight heparin (LMWH) for anticoagulation in a pregnant woman that underwent artificial mitral valve replacement 10 years prior to her pregnancy. Until she became pregnant warfarin was administered for anticoagulation, but due to the often mentioned increased risk for warfarin-induced maternal and fetal complications, at gestational week 5 the anticoagulation regimen was switched to subcutaneous application of low molecular weight heparin. At gestational week 24 our patient developed acute life-threatening pulmonary edema and hemodynamic instability due to acute mitral valve thrombosis and underwent emergency valve re-replacement with a biological porcine valve. She recovered uneventfully and gave birth to a healthy child at gestational week 35. In addition to our case presentation we review the sparse evidence in the literature regarding anticoagulation in pregnant women with mechanical heart valves and discuss the rational of different anticoagulation regimens with regards to maternal and fetal outcome. Special consideration is directed towards LMWH administration as an alternative to oral anticoagulation during pregnancy in women with mechanical heart valves.

Human CMV immediate‐early enhancer: a useful tool to enhance cell‐type‐specific expression from lentiviral vectors

Lentiviral vectors are attractive delivery tools for gene therapy, especially in terminally differentiated target cells. While restriction of gene expression to specific cell populations is of particular importance, highly efficient cell‐type‐specific gene expression after viral gene transfer so far has been hampered by low levels of transgene expression.

Clinically Established Hemostatic Scaffold (Tissue Fleece) as Biomatrix in Tissue- and Organ-Engineering Research

Various types of three-dimensional matrices have been used as basic scaffolds in myocardial tissue engineering. Many of those are limited by insufficient mechanical function, availability, or biocompatibility. We present a clinically established collagen scaffold for the development of bioartificial myocardial tissue. Neonatal rat cardiomyocytes were seeded into Tissue Fleece (Baxter Deutschland, Heidelberg, Germany). Histological and ultrastructural examinations were performed by DAPI and DiOC(18) staining and electron microscopy, respectively. Force measurements from the spontaneously beating construct were obtained. The constructs were stimulated with agents such as adrenalin and calcium, and by stretching. Passive stretch curves were obtained. Spontaneous contractions of solid bioartificial myocardial tissue (BMT), 20 x 15 x 2 mm, resulted. Contractions continued to week 12 (40% of BMTs) in culture. Histology revealed intercellular and also cell-fibril junctions. Elasticity was similar to that of native rat myocardium. Contractile force increased after topical administration of Ca(2+) and adrenaline. Stretch led to the highest levels of contractile force. In summary, bioartificial myocardial tissue with significant in vitro longevity, spontaneous contractility, and homogeneous cell distribution was produced using Tissue Fleece. Tissue Fleece constitutes an effective scaffold to engineer solid organ structures, which could be used for repair of congenital defects or replacement of diseased tissue.

Predictive Risk Factors for Patients With Cirrhosis Undergoing Heart Surgery

BACKGROUND Empiric experiences suggest higher mortality and complication risk for patients with cirrhosis of the liver after cardiac surgery. However, cirrhosis is not considered a risk factor in either the EuroSCORE or The Society of Thoracic Surgeons score. We report a large single-center experience of patients with cirrhosis undergoing cardiac surgery with extracorporeal circulation and aimed to evaluate the severity of cirrhosis as a predictor of outcome. METHODS During 2001 and 2011, we operated on 109 consecutive patients (average age, 64 years; 82 male) diagnosed for cirrhosis with cardiopulmonary bypass for different indications. Thirty-day mortality and long-term mortality were set as primary study end points. RESULTS Thirty-day mortality was 26%, and 5-year survival was 19%. Patients categorized as Child-Turcotte-Pugh (CHILD) C (n=6; 67% 30-day survival; 0% 5-year survival) and B (n=30; 60%; 5%) had worse 30-day and 5-year survival compared with patients categorized as CHILD A (n=73; 80%; 25%). For 30-day mortality, preoperative EuroSCORE (p=0.015), model for end-stage liver disease (MELD) score (p=0.006), albumin (p=0.023), total protein (p=0.01), and myocardial infarction (p=0.049) revealed significant differences between survivors and nonsurvivors. Multivariate logistic regression identified only MELD score (odds ratio [OR], 1.12; 95% confidence interval [CI], 1.03 to 1.23; p=0.011) and total protein (OR, 0.97; 95% CI, 0.95 to 1; p=0.049) were connected with increased 30-day mortality. Cox regression analysis revealed EuroSCORE (OR, 1.02; 95% CI, 1.01 to 1.03; p<0.0001) and MELD (OR, 1.06; 95% CI, 1.01 to 1.12; p=0.016) predicting the overall mortality. Receiver operating characteristic analysis indicated significant predictive power of MELD (p=0.001) and EuroSCORE (p=0.027) for 30-day mortality. CONCLUSIONS Patients with cirrhosis undergoing heart surgery with extracorporeal circulation have a poor prognosis. Several preoperative factors are related to outcome. EuroSCORE and MELD score may help to evaluate operation risk and indication.