Alpha-cardiac Myosin Heavy Chain Research Articles

TBX 5 gene mutation analysis among Tanzanian children with congenital heart diseases using high-resolution melting assays

Early cardiac development is governed by transcription factor genes. TBX5, a T-box transcription factor gene, plays an important role in the development of the second heart field during cardiac septation by promoting cell cycle progression through the enhancement of Cdk6 and hedgehog signaling pathways. TBX5 binds to the promoter region of genes, enhancing the expression of alpha cardiac myosin heavy chain 6 (MYH6), which is a predominant isoform found in human cardiac tissue. TBX5 gene mutations are postulated to cause congenital heart diseases. A casecontrol TBX5 mutational analysis was performed to provide insight into the etiology of sporadic congenital heart diseases in our setting. We used a magnetic induction cycler (mic-PCR), which is a next-generation tool for polymerase chain reaction-high resolution melting assays, to detect mutations in children with sporadic isolated congenital heart diseases. A retrospective casecontrol study was conducted at the Jakaya Kikwete Cardiac Institute. The peripheral blood samples were collected, and DNA was extracted using the Quick-DNA Miniprep Kit. The primers were designed using Primer 3 software, validated using the program BLAST, and checked for hairpin and homo-hetero-dimerization using the IDT oligo analyzer. Real-time polymerase chain reaction (PCR)-high-resolution melting assays for screening TBX5 gene mutations were done using a magnetic induction cycler. We found two (2) TBX5 mutations in exon 5, among patients with Atrial-Ventral Septal Defects (ASVD) and Atrial-Septal Defects (ASD) and none among controls. TBX5 exon 5 is a molecular hotspot for isolated congenital heart diseases.

FUNDC1 is Essential for Maintaining Cardiac Function During Prolonged Fasting

Mitochondrial quality control via mitophagy is necessary to ensure proper metabolic functioning. However, various heart diseases have been associated with mitochondrial dysfunction and thus linked to increased mortality. Our past studies have shown a time-dependent differential effect of fasting on cardiac mitophagy. Specifically, one-day fasting accelerated mitophagy flux in the mouse heart, whereas two-day fasting actually reduced mitophagy flux, which was associated with the impairment of cardiac function. However, it remains unclear if the reduced mitophagy was responsible for the impaired cardiac function. In the present study, we determined the functional role of mitophagy in the heart during fasting using mice lacking FUNDC1, a mitochondrial outer-membrane protein that serves as a mitophagy receptor. We demonstrated that a two-day fasting impaired cardiac function in wild-type mice, which was exacerbated in FUNDC1 knockout (KO) mice as determined by echocardiography and hemodynamic measurements. This suggests that FUNDC1 or FUNDC1-mediated mitophagy was essential for maintaining cardiac function during fasting. Additionally, the total ATP levels in the hearts showed no dramatic difference between fasted wild type mice and FUNDC1 KO mice, suggesting that the exacerbated cardiac function in FUNDC1 KO mice was likely unrelated to ATP production. Interestingly, FUNDC1 deficiency reduced the expression of cardiac alpha myosin heavy chain (α-MHC) but increased that of β-MHC, a gene that normally expresses during fetal development stage and has the lowest contractile capability among the 3 MHC isoforms. Thus, the altered expression of α-MHC and β-MHC might have contributed to the more severe cardiac dysfunction in FUNDC1 KO mice during fasting.

Generation of electrophysiologically functional cardiomyocytes from mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells can efficiently differentiate into the three germ layers similar to those formed by differentiated embryonic stem (ES) cells. This provides a new source of cells in which to establish preclinical allogeneic transplantation models. Our iPS cells were generated from mouse embryonic fibroblasts (MEFs) transfected with the Yamanaka factors, the four transcription factors (Oct4, Sox2, Klf4 and c-Myc), without antibiotic selection or MEF feeders. After the formation of embryoid bodies (EBs), iPS cells spontaneously differentiated into Flk1-positive cardiac progenitors and cardiomyocytes expressing cardiac-specific markers such as alpha sarcomeric actinin (α-actinin), cardiac alpha myosin heavy chain (α-MHC), cardiac troponin T (cTnT), and connexin 43 (CX43), as well as cardiac transcription factors Nk2 homebox 5 (Nkx2.5) and gata binding protein 4 (gata4). The electrophysiological activity of iPS cell-derived cardiomyocytes (iPS-CMs) was detected in beating cell clusters with optical mapping and RH237 a voltage-sensitive dye, and in single contracting cells with patch-clamp technology. Incompletely differentiated iPS cells formed teratomas when transplanted into a severe combined immunodeficiency (SCID) mouse model of myocardial infarction. Our results show that somatic cells can be reprogrammed into pluripotent stem cells, which in turn spontaneously differentiate into electrophysiologically functional mature cardiomyocytes expressing cardiac-specific makers, and that these cells can potentially be used to repair myocardial infarction (MI) in the future.

Диференціювання індукованих плюрипотентних стовбурових клітин в кардіоміоцити в суспензійній культурі з постійним перемішуванням та в спеціальних планшетах з мікролунками

In order to enhance the differentiation of induced pluripotent cells into cardiomyocytes, we compared two methods of embryoid bodies formation: differentiation in rotating suspension culture and formation of embryoid bodies from a predetermined number of pluripotent stem cells in microwells of AggreWell plates. We used transgenic murine induced pluripotent stem cell line AT25. Cell line expressed IRES-flanked enhanced green fluorescent protein (eGFP) under the control of cardiac alpha myosin heavy chain promoter (αMHC). We applied flow cytometry and fluorescence microscopyin order to test the efficiency of differentiation processes. Thus, differentiation of pluripotent stem cells in AggreWell plates without adding differentiation factors was more effective than differentiation in rotating suspension culture. However, we obtained the most amounts of cardiomyocytes on the 11-th day in rotating suspension culture with ascorbic acid, after we applied dorsomorfin, DMSO, ascorbic acid, G-CSF with the above-mentioned methods. The amount of GFP + cells was 2,71 ± 0,07%.

Autoimmunity in viral myocarditis

To review how autoimmunity is induced in viral myocarditis. Clinical and experimental myocarditis follows microbial infections, but autoimmunity to cardiac antigens leads to heart failure since infected myocytes are sparse and virus clearance is rapid. In mice, CD4+ T cells specific for cardiac alpha myosin heavy chain (αMYHC) cause myocarditis and mice tolerized to αMYHC are protected from virus challenge proving pathogenesis depends upon autoimmunity. Most importantly, multiple microbes share the same mimicking epitope with αMYHC. Serial infections with very different microbes could result in memory responses to the shared epitope leading to aggressive and severe heart failure. A similar phenomenon may explain autoimmune diseases with suspected infectious causes, where specific pathogens have not been identified. Production of the relevant cardiac epitope for antigen presentation requires more than myosin release from dead myocytes. Otherwise, myocarditis would commonly follow myocardial infarcts. The inherent nature of the innate immune response associated with viral infections in the heart is crucial to cardiac epitope expression. Antigenic mimicry between microbes and cardiac proteins causes autoimmunity in myocarditis. Characteristics of innate immunity associated with cardiac infection determine relevant epitope expression (cryptic epitopes).

Abstract P148: Urotensin II Impairs Cardiac Functions and Proliferation of Cardiac Side Population Cells in Mice During Chronic Pressure Overload.

Urotensin II (UII), a potent vasoactive “somatostatin-like” peptide, has been known to play a role in cardiovascular diseases including cardiac hypertrophy and heart failure. But its effect on cardiac side population cells (CSPs), one of somatic stem cells in the heart potentially participating in cardiac protection after injury, is unclear. The present study was therefore conducted to examine influences of UII on the differentiation, proliferation and function of CSPs. CSPs were isolated from neonatal rat hearts by fluorescence-activated cell sorting (FACS) and cultured in the absence or at the presence of UII. The expressions of alpha-cardiac myosin heavy chain, alpha-smooth muscle actin and Von Willebrand factor at mRNAs and protein levels were analyzed by reverse transcriptional PCR (RT-PCR) and immunofluoresence to evaluate the differentiation of CSPs into cardiomyocytes, smooth muscle cells and endothelial cells, respectively. The proliferation of CSPs was assessed by Luminescent Cell Viability Assay. The influence of UII on the proliferation of CSPs in vivo was also evaluated by FACS. Our results revealed that UII did inhibit the proliferation of CSPs through up-regulation of phosphorylated c-Jun N-terminal protein kinase (JNK), although it didn’t affect the differentiation of cultured CSPs into cardiaomyocytes, smooth muscle cells and endothelial cells. In vivo experiments also showed that injection of UII reduced the number of CSPs and impaired cardiac functions compared with vehicle injection in mice subjected to a chronic pressure overload, and an UII antagonist urantide induced a preserved cardiac function with an increased number of CSPs. These data indicate that UII reduces the number of CSPs by inhibiting the proliferation of CSPs possibly through increase of JNK phosphorylation, and blockage of UII may be a useful strategy for cardiac protection in the hypertensive.

Increased Cardiac Alpha-Myosin Heavy Chain in Left Atria and Decreased Myocardial Insulin-Like Growth Factor (IGF-I) Expression Accompany Low Heart Rate in Hibernating Grizzly Bears

Grizzly bears (Ursus arctos horribilis) tolerate extended periods of extremely low heart rate during hibernation without developing congestive heart failure or cardiac chamber dilation. Left ventricular atrophy and decreased left ventricular compliance have been reported in this species during hibernation. We evaluated the myocardial response to significantly reduced heart rate during hibernation by measuring relative myosin heavy-chain (MyHC) isoform expression and expression of a set of genes important to muscle plasticity and mass regulation in the left atria and left ventricles of active and hibernating bears. We supplemented these data with measurements of systolic and diastolic function via echocardiography in unanesthetized grizzly bears. Atrial strain imaging revealed decreased atrial contractility, decreased expansion/reservoir function (increased atrial stiffness), and decreased passive-filling function (increased ventricular stiffness) in hibernating bears. Relative MyHC-α protein expression increased significantly in the atrium during hibernation. The left ventricle expressed 100% MyHC-β protein in both groups. Insulin-like growth factor (IGF-I) mRNA expression was reduced by ∼50% in both chambers during hibernation, consistent with the ventricular atrophy observed in these bears. Interestingly, mRNA expression of the atrophy-related ubiquitin ligases Muscle Atrophy F-box (MAFBx) and Muscle Ring Finger 1 did not increase, nor did expression of myostatin or hypoxia-inducible factor 1α (HIF-1α). We report atrium-specific decreases of 40% and 50%, respectively, in MAFBx and creatine kinase mRNA expression during hibernation. Decreased creatine kinase expression is consistent with lowered energy requirements and could relate to reduced atrial emptying function during hibernation. Taken together with our hemodynamic assessment, these data suggest a potential downregulation of atrial chamber function during hibernation to prevent fatigue and dilation due to excessive work against an optimally filled ventricle, a response unpredicted by the Frank-Starling mechanism.

A Family of microRNAs Encoded by Myosin Genes Governs Myosin Expression and Muscle Performance

Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.

Genetic selection system allowing monitoring of myofibrillogenesis in living cardiomyocytes derived from mouse embryonic stem cells

Embryonic stem (ES) cell-derived cardiomyocytes recapitulate cardiomyogenesis in vitro and are a potential source of cells for cardiac repair. However, this requires enrichment of mixed populations of differentiating ES cells into cardiomyocytes. Toward this goal, we have generated bicistronic vectors that express both the blasticidin S deaminase (bsd) gene and a fusion protein consisting of either myosin light chain (MLC)-3f or human alpha-actinin 2A and enhanced green fluorescent protein (EGFP) under the transcriptional control of the alpha-cardiac myosin heavy chain (alpha-MHC) promoter. Insertion of the DNase I-hypersensitive site (HS)-2 element from the beta-globin locus control region, which has been shown to reduce transgene silencing in other cell systems, upstream of the transgene promoter enhanced MLC3f-EGFP gene expression levels in mouse ES cell lines. The alpha-MHC-alpha-actinin-EGFP, but not the alpha-MHC-MLC3f-EGFP, construct resulted in the correct incorporation of the newly synthesized fusion protein at the Z-band of the sarcomeres in ES cell-derived cardiomyocytes. Exposure of embryoid bodies to blasticidin S selected for a relatively pure population of cardiomyocytes within 3 days. Myofibrillogenesis could be monitored by fluorescence microscopy in living cells due to sarcomeric epitope tagging. Therefore, this genetic system permits the rapid selection of a relatively pure population of developing cardiomyocytes from a heterogeneous population of differentiating ES cells, simultaneously allowing monitoring of early myofibrillogenesis in the selected myocytes.

Neuregulin-1 promotes cardiomyocyte differentiation of genetically engineered embryonic stem cell clones

Embryonic stem (ES) cell-derived cardiomyocytes (ESCMs) must be specifically purified in order to prevent teratoma formation, and this confusing issue has hampered their clinical application. We therefore investigated a technique to generate pure labeled ESCMs for possible use in cardiac repair. We generated transgenic ES cell lines expressing enhanced green fluorescent protein (EGFP) under the transcriptional control of the alpha-cardiac myosin heavy chain (alpha-MHC) promoter. Differentiated EGFP-positive ES cells displayed characteristics of CMs. Furthermore, neuregulin-1 (NRG-1) upregulated the expression of the cardiac-restricted transcription factors Nkx2.5 and GATA-4, as well as differentiated CM factors (alpha-MHC, beta-MHC). Immunohistochemistry demonstrated that NRG-1 increased expression of cardiac-specific troponin T in the beating foci of the embryoid bodies. This work revealed a potential method for specifically labeling and enriching ESCMs by combining genetically-engineered ES cell clones and exogenous growth factor treatment.

Crossveinless-2 Controls Bone Morphogenetic Protein Signaling during Early Cardiomyocyte Differentiation in P19 Cells

Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMP-binding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of down-stream targets Smad1/5/8 protein and Id1 gene, whereas treatment with Cv2 conversely inhibited Smad1/5/8 activation and Id1 expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smad1/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Id1 and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2-kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis.

Identification of a putative shared epitope between Coxsackie virus B4 and alpha cardiac myosin heavy chain

Molecular mimicry is an important postulated mechanism for autoimmunity in viral myocarditis. The 356-1 monoclonal antibody neutralizes Coxsackie virus B4 by binding to the VP1 protein and cross-reacts with mouse alpha cardiac myosin heavy chain. We used this monoclonal antibody to screen a lambda gt11 expression library made from CD-1 mouse hearts. Of the 48 positive plaques/10(6) recombinant phages examined, 14 of the strongest-reacting clones were purified for additional studies. The inserts were amplified by polymerase chain reaction and the amplified products ranged from about 150 to 1400 bp in size. Northern hybridization using these inserts demonstrated that 11 out of 14 reacted with a message equivalent to that of cardiac myosin in size. Additional Southern hybridization studies suggested that these 11 inserts contained overlapping sequences in the light meromyosin fragment of cardiac myosin. Sequence analysis confirmed that these 11 independent, recombinant clones contained a common sequence representing amino acid residues 1299-1647. Within this fragment only one isoform-specific site matched the observed reactivity pattern of 356-1 among hearts from various species. Thus, we were able to identify a putative shared epitope represented by residues 1632-1647.

MCP-1 induces cardioprotection against ischaemia/reperfusion injury: role of reactive oxygen species

Monocyte chemoattractant protein-1 (MCP-1: CCL2) has been demonstrated to be involved in the pathophysiology of ischaemic heart disease; however, the precise role of MCP-1 in ischaemia/reperfusion (I/R) injury is controversial. Here, we investigated the role of cardiac MCP-1 expression on left ventricular (LV) dysfunction after global I/R in Langendorff-perfused hearts isolated from transgenic mice expressing the mouse JE-MCP-1 gene under the control of the alpha-cardiac myosin heavy chain promoter (MHC/MCP-1 mice). In vitro experiments showed that MCP-1 prevented the apoptosis of murine neonatal cardiomyocytes after hypoxia/reoxygenation. I/R significantly increased the mRNA expression of MCP-1 in the Langendorff-perfused hearts of wild-type mice. Cardiac MCP-1 overexpression in the MHC/MCP-1 mice improved LV dysfunction after I/R without affecting coronary flow; in particular, it ameliorated LV diastolic pressure after reperfusion. This improvement was independent of both sarcolemmal and mitochondrial K(ATP) channels. Cardiac MCP-1 overexpression prevented superoxide generation in the I/R hearts, and these hearts showed decreased expression of the NADPH oxidase family proteins Nox1, gp91phox, and Nox3 compared with the hearts of wild-type mice. Further, superoxide dismutase activity in the hearts of MHC/MCP-1 mice was significantly increased compared with that in the hearts of wild-type mice. These findings suggest that cardiac MCP-1 prevented LV dysfunction after global I/R through a reactive oxygen species-dependent but K(ATP) channel-independent pathway; this provides new insight into the beneficial role of MCP-1 in the pathophysiology of ischaemic heart diseases.

Sex-Defined T-Cell Responses to Cardiac Self Determine Differential Outcomes of Murine Dilated Cardiomyopathy

Idiopathic dilated cardiomyopathy (DCM) is a disease of putative autoimmune origin that kills males at a twofold to threefold greater frequency than females. The reasons underlying these differential outcomes may be related to sex-divergent self-recognition. Here we examined sex-specific autoimmune responses to cardiac self and their impact on DCM development. We found that males immunized to the p406-425 peptide derived from mouse cardiac alpha-myosin heavy chain preferentially develop a predominant Th17 lineage response that provides sustained T-cell memory and a high DCM incidence whereas females preferentially develop a predominant Th1 lineage response that becomes anergized to cardiac self resulting in compensatory protection against DCM. The distinct sex-defined disease phenotypes are interchangeable after in vivo manipulation of Th1 (interleukin-2) and Th17 (interleukin-17) cytokines. Our study shows that male and female SWXJ mice differentially respond to cardiac self in ways that lead to distinct autoimmune outcomes and implies that optimized therapy for autoimmunity may require consideration of the qualitatively different ways that males and females engage self.

Ligand-dependent activation of ER lowers blood pressure and attenuates cardiac hypertrophy in ovariectomized spontaneously hypertensive rats

The biological effects of oestrogens are mediated by two different oestrogen receptor (ER) subtypes, ERalpha and ERbeta, which might play different, redundant, or opposing roles in cardiovascular disease. Previously, we have shown that the selective ERalpha agonist 16alpha-LE2 improves vascular relaxation, attenuates cardiac hypertrophy, and increases cardiac output without lowering elevated blood pressure in spontaneously hypertensive rats (SHR). Because ERbeta-deficient mice exhibit elevated blood pressure and since the ERbeta agonist 8beta-VE2 attenuated hypertension in aldosterone-salt-treated rats, we have now tested the hypothesis that the isotype-selective ERbeta agonist 8beta-VE2 might be capable of lowering elevated blood pressure in ovariectomized SHR. Treatment of ovariectomized SHR with 8beta-VE2 for 12 weeks conferred no uterotrophic effects but lowered elevated systolic blood pressure (-38 +/- 5 mmHg, n = 31, P < 0.001 vs. placebo) as well as peripheral vascular resistance (-31.3 +/- 4.6%, P < 0.001 vs. placebo). 8beta-VE2 enhanced aortic ERbeta expression (+75.7 +/- 7.1%, P < 0.01 vs. placebo), improved NO-dependent vasorelaxation, augmented phosphorylation of the vasodilator-stimulated phosphoprotein in isolated aortic rings (P < 0.05 vs. placebo), increased cardiac output (+20.4 +/- 2.5%, P < 0.01 vs. placebo), and attenuated cardiac hypertrophy (-22.2 +/- 3.2%, p < 0.01 vs. placebo). 8beta-VE2, in contrast to oestradiol, did not enhance cardiac alpha-myosin heavy chain expression. Ligand-dependent activation of ERbeta confers blood pressure lowering effects in SHR that are superior to those of 17beta-estradiol or the ERalpha agonist 16alpha-LE2 and attenuates cardiac hypertrophy primarily by a reduction of cardiac afterload without promoting uterine growth.

Abstract 1213: Cardiac Overexpression of Epac1 in Transgenic Mice Protects Heart from Lipopolysaccharide-induced Cardiac Dysfunction and Inhibits JAK-STAT Pathway

The sympathetic nervous system and proinflammatory cytokines are believed to play independent roles in the pathophysiology of heart failure. However, the recent identification of Epac (exchange protein activated by cyclic AMP), a new cyclic AMP-binding protein that directly activates Rap1, have implicated that there may be a potential cross talk between the sympathetic and cytokine signals. In order to examine the role of Epac in cytokine signal to regulate cardiac function, we have generated transgenic mice expressing the human Epac1 gene under the control of alpha-cardiac myosin heavy chain promoter (Epac1-TG), and examined their response in lipopolysaccharide (LPS)-induced cardiac dysfunction, a well established model for sepsis-induced cardiac dysfunction. Sepsis-induced cardiac dysfunction results from the production of proinflammatory cytokines. At baseline, left ventricular ejection fraction (LVEF) was similar (TG vs. NTG, 67±1.7 vs. 69±2.1%, n =7–9). The degree of cardiac hypertrophy (LV(mg)/tibia(mm)) was also similar at 3 months old (TG vs. NTG 4.0±0.1 vs. 4.2±0.1, n =5–6), but it became slightly but significantly greater in Epac1-TG at 5 month old (TG vs. NTG 4.9±0.1 vs. 4.4±0.1, p&lt; 0.05, n =5–7). LPS (5mg/kg) elicited a significant and robust reduction of LVEF in both Epac1-TG and NTG, but the magnitude of this decrease was much less in Epac1-TG at 6 hr after injection (TG vs. NTG 48±2.4 vs. 57±1.8%, p&lt; 0.01, n =6–9). At 24 hr after injection, cardiac function was restored to the baseline in both Epac1-TG and NTG. We also examined the activation of JAK-STAT pathway at 24 hr after injection. The tyrosine phosphorylation of STAT1 (Tyr701) and STAT3 (Tyr705) in LV, which is an indicator of STAT activation, was reduced to a greater degree in Epac1-TG by 31±8.8% ( p&lt; 0.05, n =4) and 29±5.9% ( p&lt; 0.05, n =7), respectively, relative to that in NTG. Taken together, Epac1 protects the heart from the cytokine-induced cardiac dysfunction, at least in part, through the inhibition of the JAK-STAT pathway, suggesting the beneficial role played by sympathetic signal to antagonize proinflammatory cytokine signal in heart failure.

Cardiomyocyte expression of PPARγ leads to cardiac dysfunction in mice

Three forms of PPARs are expressed in the heart. In animal models, PPARgamma agonist treatment improves lipotoxic cardiomyopathy; however, PPARgamma agonist treatment of humans is associated with peripheral edema and increased heart failure. To directly assess effects of increased PPARgamma on heart function, we created transgenic mice expressing PPARgamma1 in the heart via the cardiac alpha-myosin heavy chain (alpha-MHC) promoter. PPARgamma1-transgenic mice had increased cardiac expression of fatty acid oxidation genes and increased lipoprotein triglyceride (TG) uptake. Unlike in cardiac PPARalpha-transgenic mice, heart glucose transporter 4 (GLUT4) mRNA expression and glucose uptake were not decreased. PPARgamma1-transgenic mice developed a dilated cardiomyopathy associated with increased lipid and glycogen stores, distorted architecture of the mitochondrial inner matrix, and disrupted cristae. Thus, while PPARgamma agonists appear to have multiple beneficial effects, their direct actions on the myocardium have the potential to lead to deterioration in heart function.

Nebulin isoforms of extraocular muscle

The extraocular muscles (EOMs), which are responsible for reflexive and voluntary eye movements, have many unique biochemical, physiological, and ultrastructural features that set them apart from other skeletal muscles. For example, rodent EOMs lack M-lines and express EOM-specific myosin heavy chain (MYH13) and alpha-cardiac myosin heavy chain. Recent gene-expression profiling studies indicate the presence of other cardiac-specific proteins in adult EOMs. This interesting mixture of myofibrillar and cytoskeletal proteins poses the questions as to whether nebulette, as opposed to nebulin, might be expressed in EOM, and what isoforms of titin are expressed in the EOM. We have performed gel electrophoresis and immunological analyses to determine the titin and nebulin isoforms expressed in the EOM. We have found that the mass of the titin isoforms expressed in the EOM most closely resemble those found in the skeletal muscles tested, viz., the soleus and extensor digitorum longus (EDL). We also demonstrate that, although the EOM expresses cardiac isoforms of myosin, it does not express nebulette and contains a nebulin isoform with a mass consistent with that found in the prototypical fast hindlimb muscle EDL.

Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function.

Dilated cardiomyopathy (DCM) leads to heart failure, a leading cause of death in industrialized nations. Approximately 30% of DCM cases are genetic in origin, with some resulting from point mutations in cardiac myosin, the molecular motor of the heart. The effects of these mutations on myosin's molecular mechanics have not been determined. We have engineered two murine models characterizing the physiological, cellular, and molecular effects of DCM-causing missense mutations (S532P and F764L) in the alpha-cardiac myosin heavy chain and compared them with WT mice. Mutant mice developed morphological and functional characteristics of DCM consistent with the human phenotypes. Contractile function of isolated myocytes was depressed and preceded left ventricular dilation and reduced fractional shortening. In an in vitro motility assay, both mutant cardiac myosins exhibited a reduced ability to translocate actin (V(actin)) but had similar force-generating capacities. Actin-activated ATPase activities were also reduced. Single-molecule laser trap experiments revealed that the lower V(actin) in the S532P mutant was due to a reduced ability of the motor to generate a step displacement and an alteration of the kinetics of its chemomechanical cycle. These results suggest that the depressed molecular function in cardiac myosin may initiate the events that cause the heart to remodel and become pathologically dilated.

Bone Marrow Cells Transdifferentiate to Cardiomyocytes When Introduced into the Embryonic Heart

Since rates of cardiomyocyte generation in the embryo are much higher than within the adult, we explored whether the embryonic heart would serve as useful experimental system for examining the myocardial potential of adult stem cells. Previously, we reported that the long-term culturing of adult mouse bone marrow produced a cell population that was both highly enriched for macrophages and cardiac competent. In this study, the myocardial potential of this cell population was analyzed in greater detail using the embryonic chick heart as recipient tissue. Experiments involving the co-incubation of labeled bone marrow cells with embryonic heart tissue showed that bone marrow (BM) cells incorporated into the myocardium and immunostained for myocyte proteins. Reverse transcription-polymerase chain reaction analysis demonstrated that the heart tissue induced bone marrow cells to express the differentiated cardiomyocyte marker alpha-cardiac myosin heavy chain. The cardiomyocyte conversion of the bone marrow cells was verified by harvesting donor cells from mice that were genetically labeled with a myocardial-specific beta-galactosidase reporter. Embryonic hearts exposed to the transgenic bone marrow in culture exhibited significant numbers of beta-galactosidase-positive cells, indicating the presence of bone marrow-derived cells that had converted to a myocardial phenotype. Furthermore, when transgenic mouse BM cells were injected into living chick embryos, donor cells incorporated into the developing heart and exhibited a myocardial phenotype. Immunofluorescence analysis demonstrated that donor BM cells exhibiting myocyte markers contained only nuclei from mouse cells, indicating that differentiation and not cell fusion was the predominant mechanism for the acquisition of a myocyte phenotype. These data confirm that adult mouse bone marrow contain cells with the ability to form cardiomyocytes. In addition, the predominance of the macrophage phenotype within the donor bone marrow cell population suggests that transdifferentiation of immune response cells may play a role in cellular regeneration in the adult.