Rafeeq P.H. Ahmed

Dilated Cardiomyopathy Mutant Tropomyosin Mice Develop Cardiac Dysfunction With Significantly Decreased Fractional Shortening and Myofilament Calcium Sensitivity

Mutations in striated muscle &agr;-tropomyosin (&agr;-TM), an essential thin filament protein, cause both dilated cardiomyopathy (DCM) and familial hypertrophic cardiomyopathy. Two distinct point mutations within &agr;-tropomyosin are associated with the development of DCM in humans: Glu40Lys and Glu54Lys. To investigate the functional consequences of &agr;-TM mutations associated with DCM, we generated transgenic mice that express mutant &agr;-TM (Glu54Lys) in the adult heart. Results showed that an increase in transgenic protein expression led to a reciprocal decrease in endogenous &agr;-TM levels, with total myofilament TM protein levels remaining unaltered. Histological and morphological analyses revealed development of DCM with progression to heart failure and frequently death by 6 months. Echocardiographic analyses confirmed the dilated phenotype of the heart with a significant decrease in the left ventricular fractional shortening. Work-performing heart analyses showed significantly impaired systolic, and diastolic functions and the force measurements of cardiac myofibers revealed that the myofilaments had significantly decreased Ca2+ sensitivity and tension generation. Real-time RT-PCR quantification demonstrated an increased expression of &bgr;-myosin heavy chain, brain natriuretic peptide, and skeletal actin and a decreased expression of the Ca2+ handling proteins sarcoplasmic reticulum Ca2+-ATPase and ryanodine receptor. Furthermore, our study also indicates that the &agr;-TM54 mutation decreases tropomyosin flexibility, which may influence actin binding and myofilament Ca2+ sensitivity. The pathological and physiological phenotypes exhibited by these mice are consistent with those seen in human DCM and heart failure. As such, this is the first mouse model in which a mutation in a sarcomeric thin filament protein, specifically TM, leads to DCM.

Inherited prothrombotic defects in Budd-Chiari syndrome and portal vein thrombosis: a study from North India.

We studied 57 patients with Budd-Chiari syndrome (BCS) and 48 with portal vein thrombosis (PVT) for underlying inherited prothrombotic defects such as protein C, protein S, and antithrombin III deficiencies. Genetic mutations for factor V Leiden, prothrombin gene 20210A, and methyltetrahydrofolate reductase (MTHFR) C677T were studied in 29 patients in each group. Inherited prothrombotic defects were detected in 16 (28%) of 57 patients with BCS and 7 (15%) of 48 patients with PVT. Factor V Leiden mutation was the most common prothrombotic defect in BCS (5/29 [17%]) followed by protein C deficiency (7/57 [12%]) and protein S deficiency (4/57 [7%]), whereas in PVT, protein C deficiency was the most common inherited prothrombotic defect (4/48 [8%]) followed by protein S deficiency (2/48 [4%]). The factor V Leiden mutation was detected in only 1 (3%) of 29 cases of PVT. The heterozygous MTHFR C677T mutation was detected in 7 (24%) of 29 patients with BCS and 6 (21%) of 29 patients with PVT. Antithrombin III deficiency, homozygous MTHFR C677T mutation, and prothrombin G20210A mutation were not detected in any patients.

Transcriptional profiling of young and old mesenchymal stem cells in response to oxygen deprivation and reparability of the infarcted myocardium

Most clinical studies have used autologous bone marrow (BM) stem cells for myocardial regeneration in elderly patients. We hypothesize that aging impairs the survival and differentiation potential of BM stem cells thus limiting their therapeutic efficacy. BM-derived MSCs from young ((Yng)MSCs; 8-12 weeks) and old ((Old)MSCs; 24-26 months) rats were purified and assessed for their responsiveness to anoxia and reparability of infarcted heart. Higher expression of angiogenic growth factors was observed by (Yng)MSCs under anoxia as compared to (Old)MSCs, cultured either alone or in co-culture ((Co-old)MSCs) with (Yng)MSCs. Likewise, (Yng)MSCs were more tolerant to apoptotic stimuli and showed higher ability to form tubular structures during in vitro Matrigel assay as compared to (Old)MSCs and (Co-old)MSCs with a possible role of p21 and p27 as contributory survival factors. For in vivo studies, acute myocardial infarction model was developed in Fischer-344 rats (n=38). The animals were grouped to receive 70 microl basal DMEM without cells (group 1) or containing 2 x 10(6)(Yng)MSCs (PKH67 labeled; group 2) or (Old)MSCs (PKH26 labeled; group 3) and mixture of (Yng)MSCs + (Old)MSCs (1 x 10(6) cells each; group 4). Histological studies revealed that by day 7, (Yng)MSCs showed elongated morphology with orientation similar to the host muscle architecture. Electron microscopy and confocal imaging after fluorescent immunostaining showed superior angiomyogenic potential of (Yng)MSCs. Echocardiography showed significantly preserved heart function indices in the animals transplanted with (Yng)MSCs. Aging impairs the responsiveness of (Old)MSCs to anoxia and their differentiation potential. (Yng)MSCs fail to alter the survival of (Old)MSCs under in vitro as well as in vivo conditions. It is therefore concluded that transplantation of stem cells from young donors would be a better option for heart cell therapy in future clinical studies.

Sonic Hedgehog Gene Delivery to the Rodent Heart Promotes Angiogenesis via iNOS/Netrin-1/PKC Pathway

Background We hypothesized that genetic modification of mesenchymal stem cells (MSCs) with Sonic Hedgehog (Shh) transgene, a morphogen during embryonic development and embryonic and adult stem cell growth, improved their survival and angiogenic potential in the ischemic heart via iNOS/netrin/PKC pathway. Methods/Principal Findings MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid (ShhMSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in ShhMSCs which also led to increased expression of angiogenic and pro-survival growth factors in ShhMSCs. Significantly improved migration and tube formation was seen in ShhMSCs as compared to empty vector transfected MSCs (EmpMSCs). Significant upregulation of netrin-1 and iNOS was observed in ShhMSCs in PI3K independent but PKC dependent manner. For in vivo studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 µl basal DMEM without cells (group-1) or containing 1×106 EmpMSCs (group-2) and ShhMSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of ShhMSCs post engraftment. Histological studies combined with fluorescent microspheres showed increased density of functionally competent blood vessels in group-3 and group-4. Echocardiography showed significantly preserved heart function indices post engraftment with ShhMSCs in group-3 animals. Conclusions/Significance Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.

Preconditioning Promotes Survival and Angiomyogenic Potential of Mesenchymal Stem Cells in the Infarcted Heart via NF-κB Signaling

We proposed that pharmacological manipulation of mesenchymal stem cells (MSCs) with diazoxide enhanced their survival and regenerative potential via NFkappaB regulation. MSCs preconditioned ((PC)MSCs) with diazoxide and later subjected to oxidant stress with 100 micromol/L H(2)O(2) either immediately or after 24 h exhibited higher survival (p < 0.01 vs nonpreconditioned MSCs; (Non-PC)MSCs) with concomitantly increased phosphorylation of PI3K, Akt, GSK3beta (cytoplasmic), and NF-kappaB (p65) (nuclear). Akt kinase activity was determined as a function of GSK3beta activity. Pretreatment of (PC)MSCs with Wortmannin (Wt), NEMO-binding domain (NBD), or NF-kappaB (p50) siRNA abolished NF-kappaB (p65) activity. Preconditioning increased NF-kappaB-dependent elevation of secretable growth factors associated with their paracrine effects. Inhibition of PI3K activity with Wt reduced (PC)MSCs viability at both early and 24 h time-points. However, inhibition of NF-kappaB reduced viability of (PC)MSCs only at 24 h time-point. For in vivo studies, DMEM without cells (group-1) or containing 1 x 10(6) male (Non-PC)MSCs (group-2), (PC)MSCs (group-3), (PC)MSCs pretreated with Wortmannin (group-4) or NF-kappaB decoy (group-5) were transplanted in a female rat model of acute myocardial infarction. Group-3 showed highest cell survival and growth factor expression, increased angiomyogenesis, and functional improvement. We conclude that activation of NF-kappaB by preconditioning promoted (PC)MSCs survival and angiomyogenic potential in the infarcted heart.

Inherited Prothrombotic Defects in Budd-Chiari Syndrome and Portal Vein Thrombosis

We studied 57 patients with Budd-Chiari syndrome (BCS) and 48 with portal vein thrombosis (PVT) for underlying inherited prothrombotic defects such as protein C, protein S, and antithrombin III deficiencies. Genetic mutations for factor V Leiden, prothrombin gene 20210A, and methyltetrahydrofolate reductase (MTHFR) C677T were studied in 29 patients in each group. Inherited prothrombotic defects were detected in 16 (28%) of 57 patients with BCS and 7 (15%) of 48 patients with PVT. Factor V Leiden mutation was the most common prothrombotic defect in BCS (5/29 [17%]) followed by protein C deficiency (7/57 [12%]) and protein S deficiency (4/57 [7%]), whereas in PVT, protein C deficiency was the most common inherited prothrombotic defect (4/48 [8%]) followed by protein S deficiency (2/48 [4%]). The factor V Leiden mutation was detected in only 1 (3%) of 29 cases of PVT. The heterozygous MTHFR C677T mutation was detected in 7 (24%) of 29 patients with BCS and 6 (21%) of 29 patients with PVT. Antithrombin III deficiency, homozygous MTHFR C677T mutation, and prothrombin G20210A mutation were not detected in any patients.

Cardiac tumorgenic potential of induced pluripotent stem cells in an immunocompetent host with myocardial infarction

AIM Genetic reprogramming of somatic cells with stemness genes to restore their pluripotent status is being studied extensively to generate pluripotent stem cells as an alternative to embryonic stem cells. This study was designed to examine the effectiveness of skeletal myoblast-derived induced pluripotent stem cells (SkiPS) from young male Oct4/GFP transgenic mice for regeneration of the infarcted heart. METHODS & RESULTS A mouse model of permanent coronary artery ligation was developed in young female immunocompetent C57BL/6J or C57BL/6x129S4 SV/jae Oct4/GFP mice. SkiPS labeled with Q-dots (3 × 10(5) in 10 µl basal Dulbeccos modified Eagles medium) were transplanted in and around the area of infarct immediately after coronary artery ligation (n = 16) under direct vision. Control mice (n = 12) were injected with the same number of skeletal myoblasts. Histological studies documented successful engraftment of SkiPS in all the surviving animals 4 weeks later. However, six of the 16 SkiPS-transplanted (37.5%) animal hearts showed intramural teratomas, whereas no tumor growth was observed in the control mice. Q-dot-labeled donor cells were also observed at the site of tumors. Histological studies revealed that teratomas were composed of cells from all of the three embryonic germ layers. Ultra-structure studies confirmed the histological findings and showed regions with well-organized myofibrillar structures in the tumors. CONCLUSION Undifferentiated induced pluripotent stem cells should not be recommended for cardiac transplantation unless screened for specific teratogenic precursors or predifferentiated into cardiac lineage prior to transplantation.

Reprogramming of skeletal myoblasts for induction of pluripotency for tumor-free cardiomyogenesis in the infarcted heart.

Rationale: Skeletal myoblasts (SMs) with inherent myogenic properties are better candidates for reprogramming to pluripotency. Objective: To reprogram SMs to pluripotency and show that reprogrammed SMs (SiPS) express embryonic gene and microRNA profiles and that transplantation of predifferentiated cardiac progenitors reduce tumor formation. Methods and Results: The pMXs vector containing mouse cDNAs for Yamanakas quartet of stemness factors were used for transduction of SMs purified from male Oct4-GFP+ transgenic mouse. Three weeks later, GFP+ colonies of SiPS were isolated and propagated in vitro. SiPS were positive for alkaline phosphatase, expressed SSEA1, and displayed a panel of embryonic stem (ES) cell–specific pluripotency markers. Embryoid body formation yielded beating cardiomyocyte-like cells, which expressed early and late cardiac-specific markers. SiPS also had an microRNA profile that was altered during their cardiomyogenic differentiation. Noticeable abrogation of let-7 family and significant up-regulation of miR-200a-c was observed in SiPS and SiPS-derived cardiomyocytes, respectively. In vivo studies in an experimental model of acute myocardial infarction showed extensive survival of SiPS and SiPS-derived cardiomyocytes in mouse heart after transplantation. Our results from 4-week studies in DMEM without cells (group 1), SMs (group-2), SiPS (group-3), and SiPS-derived cardiomyocytes (group 4) showed extensive myogenic integration of the transplanted cells in group 4 with attenuated infarct size and improved cardiac function without tumorgenesis. Conclusions: Successful reprogramming was achieved in SMs with ES cell-like microRNA profile. Given the tumorgenic nature of SiPS, their predifferentiation into cardiomyocytes would be important for tumor-free cardiogenesis in the heart.

Factor V Leiden—the commonest molecular defect in arterial and venous thrombophilia in India

Several genetic factors are believed to predispose to thrombophilia [1,2]. These include Factor V Leiden defect, Prothrombin G20210A and MTHFR C677T gene polymorphism. FV Leiden is the most frequent molecular defect seen in thrombophilia in Caucasians. It has been found in 20% of consecutive patients with juvenile deep vein thrombosis (DVT), 3% arterial thrombosis and about 4.2% of healthy Caucasians [3–7]. Moderate hyperhomocysetinemia secondary to homozygous C-to-T substitution at nucleotide position 677 of the 5,10 MTHFR gene, has been found to underlie 19% of arterial, 11% of venous thrombophilia and 12% of controls [8]. Mutation at nucleotide position 20210 of the prothrombin gene is believed to influence the regulation of prothrombin gene expression and is associated with approximately threefold increase in risk for DVT in the West [9]. There it has been seen in 2% of arterial and 8% of venous thrombophilia and 2.1% of controls. Since no study exists from India on molecular defects underlying arterial thrombosis [10], and only one study is available, for venous thrombosis, the aim in this study was to determine the occurrence of these mutations in Indian subjects with arterial and venous thrombosis.

Mutation reports: Intron 1 and 22 inversions in Indian haemophilics

Intron 1 and 22 inversions were looked for in 80 severe haemophilia A patients in India using PCR and multiplex Long-Distance Subcycling-PCR, respectively. Intron 1 inversion was seen in 3 (3.75%) and intron 22 inversion was seen in 35 (43.75%) patients. Of severe haemophilics, 47.5% had either of these inversions. It is thus suggested that screening for inversions may be the first step in genetic testing of Indian haemophilics.