The Formation of Aberrant Collateral Vessels during Coronary Arteriogenesis in Dog Heart.

We previously reported excessive growth of collateral vessels in the dog heart during arteriogenesis induced by implantation of an ameroid constrictor around the circumflex branch of the left coronary artery. In the present study, using histology and immunocofocal microscopy, we further investigated how these aberrant collateral vessels form. By comparison with mature collateral vessels the following findings were made: perivascular space was very narrow where damage of the perivascular myocardium occurred; the neointima was very thick, resulting in a very small lumen; elastica van Gieson staining revealed the absence of the internal elastic lamina and of elastic fibers in the adventitia, but abundant collagen in the adventitia as well as in the neointima; smooth muscle cells of the neointima expressed less α-SM actin and little desmin; expression of the fibroblast growth factors aFGF, bFGF and platelet-derived growth factor (PDGF)-AB was observed mainly in the endothelial cells and abluminal region, but transforming growth factor-ß1 was only present in the adventitia and damaged myocardium; angiogenesis in the neointima was observed in some collateral vessels expressing high levels of eNOS, and cell proliferation was mainly present in the abluminal region, but apoptosis was in the deep neointima. In conclusion, these data for the first time reveal that the formation of the aberrant collateral vessels in the dog heart involves active extracellular proteolysis and a special expression profile of growth factors, eNOS, cell proliferation and apoptosis. The finding of a narrow perivascular space and perivascular myocardial damage suggests that anatomical constraint is most likely the cause for exacerbated inward remodeling in aberrant collateral vessels in dog heart.

A common MLP (muscle LIM protein) variant is associated with cardiomyopathy.

RATIONALE: We previously discovered the human 10T-->C (Trp4Arg) missense mutation in exon 2 of the muscle LIM protein (MLP, CSRP3) gene. OBJECTIVE: We sought to study the effects of this single-nucleotide polymorphism in the in vivo situation. METHODS AND RESULTS: We now report the generation and detailed analysis of the corresponding Mlp(W4R/+) and Mlp(W4R/W4R) knock-in animals, which develop an age- and gene dosage-dependent hypertrophic cardiomyopathy and heart failure phenotype, characterized by almost complete loss of contractile reserve under catecholamine induced stress. In addition, evidence for skeletal muscle pathology, which might have implications for human mutation carriers, was observed. Importantly, we found significantly reduced MLP mRNA and MLP protein expression levels in hearts of heterozygous and homozygous W4R-MLP knock-in animals. We also detected a weaker in vitro interaction of telethonin with W4R-MLP than with wild-type MLP. These alterations may contribute to an increased nuclear localization of W4R-MLP, which was observed by immunohistochemistry. CONCLUSIONS: Given the well-known high frequency of this mutation in Caucasians of up to 1%, our data suggest that (W4R-MLP) might contribute significantly to human cardiovascular disease.

Collateral vessel growth induced by femoral artery ligature is impaired by denervation.

Innervation plays an important role in development and remodeling of blood vessels. However, very little is known whether innervation is involved in arteriogenesis. In the present study, we tested the hypothesis that innervation may contribute to the process of arteriogenesis induced by ligature of femoral artery in rat/rabbit hind limb with or without denervation. We found that: (1) angiography showed more collateral vessels in the ligature side than that in ligature plus denervation side; (2) collateral vessels in denervation side was characterized by an inward remodeling; (3) in both collateral vessels (CVs) from only femoral ligature side as well as the ligature plus denervation side, ICAM-1 and VCAM-1 expression was up-regulated but increased VCAM-1 was more evident in the adventitia of collateral vessels of only femoral ligature side; (4) 7 days after surgery, in CVs from the femoral ligature side only, numerous macrophages (RAM11 positive cells) and high cell proliferation ratio (ki67 positive cells) were detected, but they were less in the denervation side. In conclusion, our data demonstrate for the first time that neural regulation is one of the factors that contributes to collateral vessel growth in rat/rabbit hind limb ischemic model by showing collateral vessel growth induced by femoral artery ligature is impaired by denervation.

Immunohistochemical study of the growth factors, aFGF, bFGF, PDGF-AB, VEGF-A and its receptor (Flk-1) during arteriogenesis.

Growth factors are viewed as main arteriogenic stimulators for collateral vessel growth. However, the information about their native expression and distribution in collateral vessels is still limited. This study was designed to profile expression of acidic and basic FGF, platelet-derived growth factor (PDGF-AB) and vascular endothelial growth factor (VEGF-A) and its receptor, fetal liver kinase-1 (Flk-1) during arteriogenesis by confocal immunofluorescence in both dog ameroid constrictor model and rabbit arteriovenous shunt model of arteriogenesis. We found that: (1) in normal arteries (NA) in dog heart, aFGF, bFGF, and PDGF-AB all were mainly expressed in endothelial cells (EC) and media smooth muscle cells (SMC), but the expression of aFGF was very weak, with those of the other two being moderate; (2) in collateral arteries (CAs), aFGF, bFGF, and PDGF-AB all were significantly upregulated (P < 0.05); they were present in all the layers of the vascular wall and were 2.1, 1.7, and 1.9 times higher than that in NA, respectively; and (3) in NA in rabbit hind limb, VEGF-A was absent, Flk-1 was only weakly present in endothelial cells, but in one week CAs VEGF-A and Flk-1 were significantly increased in both shunt and ligation sides; this was more evident in the shunt-side CAs, 2.3, and 2 times higher than that in the ligation side, respectively. In conclusion, our data demonstrate for the first time that growth factors, aFGF, bFGF, and PDGF-AB are significantly upregulated in collateral vessels in dog heart, and enhanced VEGF-A and its receptor, Flk-1, are associated with rapid and lasting increased shear stress. These findings suggest that endogenous production of growth factors could be an important factor promoting collateral vessel growth.

Activation of the integrins alpha 5beta 1 and alpha v beta 3 and focal adhesion kinase (FAK) during arteriogenesis.

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins alpha 5 beta 1 and v beta 3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins alpha 5 beta 1 and alpha v beta 3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both alpha 5 beta 1 and alpha v beta 3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was alpha 5 beta 1 and alpha v beta 3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), alpha 5 beta 1, and alpha v beta 3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress.

Expression profiling of cardiac genes in Tako-Tsubo cardiomyopathy: insight into a new cardiac entity.

Tako-Tsubo cardiomyopathy (TTC) is characterized by a transient contractile dysfunction, but its specific pathomechanism remains unknown. Thus, we performed a systematic expression profiling of genes by microarray analysis in the acute phase and after functional recovery. We studied 3 female patients presenting with TTC. Complementary RNA was isolated from left ventricular biopsies taken in the acute phase (group A) and after functional recovery (group B). It was profiled for gene expression using cDNA microarrays. Functionally related genes were determined with the Gene Set Enrichment Analysis (GSEA) bioinformatic tool. Validation of selected genes was performed by means of real-time PCR and immunohistochemistry. In group A, different functional gene sets, such as Nrf2-induced genes, triggered by oxidative stress, and protein biosynthesis were significantly overrepresented among the upregulated targets. Increased transcription of GPX1, CAT, RPS6, and eIF4E was confirmed by RT-PCR. The targets of the Akt/PKB signaling showed significant upregulation in both groups. Immunohistochemistry showed that the downstream targets NF-kappaB and BcL-X(L) are upregulated and activated. Gene sets involved in energy metabolism (oxidative phosphorylation, mitochondrial genes) showed no differences in group A but were overexpressed in group B. This study demonstrated a significant contribution of oxidative stress to the pathomechanism of TTC; it is possibly triggered by excess catecholamine. Increased protein biosynthesis and an activated cell survival cascade can be interpreted as potential compensatory mechanisms. After functional recovery, processes involved in energy metabolism play a pivotal role, thereby potentially contributing to the normalization of contractile function.

Laminin-alpha4 and integrin-linked kinase mutations cause human cardiomyopathy via simultaneous defects in cardiomyocytes and endothelial cells.

BACKGROUND: Extracellular matrix proteins, such as laminins, and endothelial cells are known to influence cardiomyocyte performance; however, the underlying molecular mechanisms remain poorly understood. METHODS AND RESULTS: We used a forward genetic screen in zebrafish to identify novel genes required for myocardial function and were able to identify the lost-contact (loc) mutant, which encodes a nonsense mutation in the integrin-linked kinase (ilk) gene. This loc/ilk mutant is associated with a severe defect in cardiomyocytes and endothelial cells that leads to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between laminin-alpha4 (Lama4), integrin, and Ilk, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy. We identified 2 novel amino acid residue-altering mutations (2828C>T [Pro943Leu] and 3217C>T [Arg1073X]) in the integrin-interacting domain of the LAMA4 gene and 1 mutation (785C>T [Ala262Val]) in the ILK gene. Biacore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin-binding capacity in case of the Pro943Leu (Kd=5+/-3 micromol/L) and Arg1073X LAMA4 (Kd=1+/-0.2 micromol/L) mutants compared with the wild-type LAMA4 protein (Kd=440+/-20 nmol/L). Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, which ultimately leads to heart failure. CONCLUSIONS: This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.

Bone marrow-derived cells contribute to infarct remodelling.

OBJECTIVE: The paradigm that cardiac myocytes are non-proliferating and terminally differentiated cells has recently been challenged by several studies reporting the ability of bone marrow-derived cells (BMC) to transdifferentiate into cardiomyocytes. However, these results are controversial and could not be reproduced by others. Therefore, we studied the contribution and potential transdifferentiation of BMC into different cell types during the remodelling process in mouse hearts with experimental myocardial infarction. METHODS: Mice (C57BL/6J) were sublethally irradiated, and BM from enhanced green fluorescent protein (eGFP)-transgenic mice was transplanted. Coronary artery ligation was performed 3 months later. The hearts were studied 7 days (n=13) and 21 days (n=12) after infarction. Immunohistochemical staining was performed using antibodies against titin, connexin 43, vimentin, SMemb alpha-smooth muscle actin, CD45, CD34, F4/80, BS-1, CD31, and eGFP. Sections were analyzed using fluorescence and confocal laser microscopy. RESULTS: Success of BM transplantation was confirmed by FACS analysis. Occlusion of the coronary artery resulted in infarct sizes of 41+/-6% of the left ventricle. CD45+/eGFP+ inflammatory cells were found frequently after 7 days and to a lesser degree after 21 days. In 25 examined hearts, only 3 eGFP-positive cardiomyocytes were found. However, numerous BMC-derived fibroblasts and myofibroblasts were found in the infarct area. BMC contributed to scar tissue neoangiogenesis but not to angiogenesis in the periinfarct and remote zones. CONCLUSION: Transdifferentiation of BMC into viable cardiomyocytes is a negligible event in normal repair processes after myocardial damage. BMC-derived fibroblasts and myofibroblasts as well as neoangiogenesis significantly contribute to post-infarction scar formation and might be important in scar tissue remodelling.

microRNA-352 regulates collateral vessel growth induced by elevated fluid shear stress in the rat hind limb.

Although collateral vessel growth is distinctly enhanced by elevated fluid shear stress (FSS), the underlying regulatory mechanism of this process remains incompletely understood. Recent studies have shown that microRNAs (miRNAs) play a pivotal role in vascular development, homeostasis and a variety of diseases. Therefore, this study was designed to identify miRNAs involved in elevated FSS-induced collateral vessel growth in rat hind limbs. A side-to-side arteriovenous (AV) shunt was created between the distal stump of one of the bilaterally occluded femoral arteries and the accompanying vein. The miRNA array profile showed 94 differentially expressed miRNAs in FSS-stressed collaterals including miRNA-352 which was down-regulated. Infusion of antagomir-352 increased the number and proliferation of collateral vessels and promoted collateral flow restoration in a model of rat hind limb ligation. In cell culture studies, the miR-352 inhibitor increased endothelial proliferation, migration and tube formation. In addition, antagomir-352 up-regulated the expression of insulin-like growth factor II receptor (IGF2R), which may play a part in the complex pathway leading to arterial growth. We conclude that enhanced collateral vessel growth is controlled by miRNAs, among which miR-352 is a novel candidate that negatively regulates arteriogenesis, meriting additional studies to unravel the pathways leading to improved collateral circulation.

Myocytes die by multiple mechanisms in failing human hearts.

We tested the hypothesis that myocyte loss in failing human hearts occurs by different mechanisms: apoptosis, oncosis, and autophagic cell death. Explanted hearts from 19 patients with idiopathic dilated cardiomyopathy (EF< or =20%) and 7 control hearts were analyzed. Myocyte apoptosis revealed by caspase-3 activation and TUNEL staining occurred at a rate of 0.002+/-0.0005% (P<0.05 versus control) and oncosis assessed by complement 9 labeling at 0.06+/-0.001% (P<0.05). Cellular degeneration including appearance of ubiquitin containing autophagic vacuoles and nuclear disintegration was present at the ultrastructural level. Nuclear and cytosolic ubiquitin/protein accumulations occurred at 0.08+/-0.004% (P<0.05). The ubiquitin-activating enzyme E1 and the ligase E3 were not different from control. In contrast, ubiquitin mRNA levels were 1.8-fold (P<0.02) elevated, and the conjugating enzyme E2 was 2.3-fold upregulated (P<0.005). The most important finding, however, is the 2.3-fold downregulation of the deubiquitination enzyme isopeptidase-T and the 1.5-fold reduction of the ubiquitin-fusion degradation system-1, which in conjunction with unchanged proteasomal subunit levels and proteasomal activity results in massive storage of ubiquitin/protein complexes and in autophagic cell death. A 2-fold decrease of cathepsin D might be an additional factor responsible for the accumulation of ubiquitin/protein conjugates. It is concluded that in human failing hearts apoptosis, oncosis, and autophagy act in parallel to varying degrees. A disturbed balance between a high rate of ubiquitination and inadequate degradation of ubiquitin/protein conjugates may contribute to autophagic cell death. Together, these different types of cell death play a significant role for myocyte disappearance and the development of contractile dysfunction in failing hearts.

Adult rat cardiac myocytes in culture: 'Second-floor' cells and coculture experiments.

OBJECTIVE: To compare adult rat cardiomyocytes in primary culture for up to 28 days with those in primary culture for 10 days plus up to 18 days in a coculture (CC) system. The phenomenon of 'second-floor' cells in primary cultures and the behaviour of CC myocytes were studied over varying times with regard to protein content and attachment to underlying cells. METHODS: Qualitative confocal microscopy and quantitation using the Imaris program (Bitplane, Switzerland) for the measurement of the fluorescence intensity in the confocal microscope were used. The protein content of actin, myosin, desmin, tubulin, titin, myomesin, cadherin and connexin was determined. Cell death was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling method for apoptosis and using propidium iodide applied to unfixed cultures to detect necrosis. RESULTS: Compared with controls, second-floor cells contained only 14% of the actin and 4.9% of the tubulin at 10 days, whereas these proteins were well preserved in CC cells. All other proteins slowly declined in second-floor cells, whereas they were still present in normal amounts in CC cells. Cell death was evident in second-floor cells but absent in CC myocytes. Cellular attachment was still evident through original in vivo adherens junctions in second-floor cells but numerous newly developed cadherin- and connexin-containing junctions were visible in CC cells. It appears from the present study that second-floor cells are mummified dead cardiomyocytes, whereas CC myocytes survive and start to dedifferentiate. CONCLUSION: The absence of actin and tubulin, together with nuclear changes, are indicators of loss of cell viability despite preservation of the cells' rod shape and cross-striation, as observed in second-floor cells. In contrast, the establishment of a CC system of cardiomyocytes results in survival and organization of a three-dimensional cellular system, which may in the future be useful for tissue engineering attempts for replacement of lost tissue after myocardial infarction.

Gap junctional remodeling by hypoxia in cultured neonatal rat ventricular myocytes.

OBJECTIVES: Altered gap junctional coupling of ventricular myocytes plays an important role in arrhythmogenesis in ischemic heart disease. Since hypoxia is a major component of ischemia, we tested the hypothesis that hypoxia causes gap junctional remodeling accompanied by conduction disturbances. METHODS: Cultured neonatal rat ventricular myocytes were exposed to hypoxia (1% O(2)) for 15 min to 5 h, connexin43 (Cx43) expression was analyzed, and conduction velocity was measured using the Micro-Electrode Array data acquisition system. RESULTS: After 15 min of hypoxia, conduction velocity was unaffected, while total Cx43, including the phosphorylated and nonphosphorylated isoforms, was increased. After 5 h of hypoxia, total Cx43 protein was decreased by 50%, while the nonphosphorylated Cx43 isoform was unchanged. Confocal analyses yielded a 55% decrease in the gap junctional Cx43 fluorescence signal, a 55% decrease in gap junction number, and a 26% decrease in size. The changes in Cx43 were not accompanied by changes in mRNA levels. The reduction in Cx43 protein levels was associated with a approximately 20% decrease in conduction velocity compared to normoxic cultures. CONCLUSIONS: Short-term hypoxia (5 h) decreases Cx43 protein and conduction velocity, thereby contributing to the generation of an arrhythmogenic substrate.

Progression from compensated hypertrophy to failure in the pressure-overloaded human heart: structural deterioration and compensatory mechanisms.

BACKGROUND: The progression of compensated hypertrophy to heart failure (HF) is still debated. We investigated patients with isolated valvular aortic stenosis and differing degrees of left ventricular (LV) systolic dysfunction to test the hypothesis that structural remodeling, as well as cell death, contributes to the transition to HF. METHODS AND RESULTS: Structural alterations were studied in LV myectomies from 3 groups of patients (group 1: ejection fraction [EF] >50%, n=12; group 2: EF 30% to 50%, n=12; group 3: EF <30%, n=10) undergoing aortic valve replacement. Control patients were patients with mitral valve stenosis but normal LV (n=6). Myocyte hypertrophy was accompanied by increased nuclear DNA and Sc-35 (splicing factor) content. ACE and TGF-beta1 were upregulated correlating with fibrosis, which increased 2.3-, 2.2-, and 3.2-fold over control in the 3 groups. Myocyte degeneration increased 10, 22, and 32 times over control. A significant correlation exists between EF and myocyte degeneration or fibrosis. Ubiquitin-related autophagic cell death was 0.5 per thousand in control and group 1, 1.05 in group 2, and 6.05 per thousand in group 3. Death by oncosis was 0 per thousand in control, 3 per thousand in group 1, and increased to 5 per thousand (groups 2 and 3). Apoptosis was not detectable in control and group 3, but it was present at 0.02 per thousand in group 1 and 0.01 per thousand in group 2. Cardiomyocyte mitosis was never observed. CONCLUSIONS: These structure-function correlations confirm the hypothesis that transition to HF occurs by fibrosis and myocyte degeneration partially compensated by hypertrophy involving DNA synthesis and transcription. Cell loss, mainly by autophagy and oncosis, contributes significantly to the progression of LV systolic dysfunction.

Matrix metalloproteinases and their tissue inhibitors in pressure-overloaded human myocardium during heart failure progression.

OBJECTIVES: We studied the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in fibrosis formation in the transition from hypertrophy to heart failure (HF) as well as the cellular source of MMPs and TIMPs. BACKGROUND: Human pressure-overloaded hearts are characterized by a significant increase in cardiac fibrosis. However, the contribution of the proteolytic/antiproteolytic system in aortic stenosis (AS) during hypertrophy progression has not yet been elucidated. METHODS: Three groups of AS patients (I: EF >50%, n = 12; II: EF 50% to 30%, n = 10; III: EF <30%, n = 12) undergoing aortic valve replacement and seven controls were studied. Tissue samples were investigated by immunoconfocal microscopy, Western blotting, and zymography. RESULTS: Quantitative analysis by immunoconfocal microscopy and Western blotting showed an upregulation of MMP-1, -2, -3, -9, -13, and -14 in group I and further increases in later stages. Tissue inhibitors of metalloproteinase-1 and -2 were enhanced and TIMP-4 was decreased in comparison to control. Gelatinolytic activity of MMP-2 significantly (p < 0.05) increased 1.2-fold (group I), 1.5-fold (group II), and 1.6-fold (group III) over control. The level of collagen I was significantly upregulated in all AS groups. Immunoconfocal microscopy showed that MMPs and TIMPs are produced predominantly by fibroblasts. The number of proliferating fibroblasts was significantly elevated during the transition to HF (0.67 n/mm(2)-control, 5.03-group III, p < 0.05). CONCLUSIONS: In human hearts a continuous turnover of the extracellular matrix occurs during the progression from compensated hypertrophy to HF that is characterized by the upregulation of MMPs and inadequate inhibition by TIMPs. The altered balance between proteolysis/antiproteolysis with accompanying proliferation of fibroblasts results in fibrosis progression.

Human hibernating myocardium is jeopardized by apoptotic and autophagic cell death.

OBJECTIVES: The aim of the present study was to objectify the loss of myocytes and the mechanism by which myocytes die in human hibernating myocardium (HHM). BACKGROUND: Intracellular degeneration, reduced cellular protein synthesis, and the replacement fibrosis contribute to structural disintegration of HHM. METHODS: In 14 patients, HHM was diagnosed by dobutamine echocardiography, radionuclide ventriculography, and thallium-201 scintigraphy. Functional recovery was documented by repeating the preoperative clinical investigations three months after successful coronary artery bypass graft surgery (CABG). During CABG, transmural biopsies were taken from the center of HHM regions and studied by electron microscopy, immunohistochemistry, the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) method, reverse transcription-polymerase chain reaction, and Western blotting. Control samples were taken from nondiseased human myocardium. RESULTS: All patients showed significant improvement or normalization of the regional function of HHM. Ubiquitin-related autophagic cell death was evident ultrastructurally by the occurrence of autophagic vacuoles, cellular degeneration, and nuclear disassembly. Ubiquitin-protein complexes were found in 0.03 +/- 0.008% (control: 0%, p < 0.005) of all myocytes. The proteasome 20S subunit/total myocytes were reduced from 63.3 +/- 9.6% in control myocardium to 36.9 +/- 8.4% in HHM. Complement-9, indicating oncosis, was found in only one of 14 biopsies. TUNEL-positive myocytes were 0.002 +/- 0.0003%. Electron microscopy showed apoptotic cells in 3 of 14 samples. However, the bcl-2/bax ratio was significantly reduced. Moreover, caspase-3 messenger ribonucleic acid was 8.5 times upregulated, and caspase-3 was activated. Cell death was absent in controls. CONCLUSIONS: In HHM, ubiquitin-related autophagic cell death and apoptosis cause a loss of myocytes. This plays an important role in progressive tissue damage and causes a reduction of the extent of functional recovery of HHM.

Weakness of a giant: mutations of the sarcomeric protein titin.

In 2002, three reports described for the first time mutations in the sarcomeric protein titin associated with dilated cardiomyopathy in humans. Despite different locations (Z-line region, Z-I transitional zone, N2B region, half A band region) all mutations resulted in heart failure. In addition, an N2B mutation was found in zebrafish embryos with ventricular dilatation and cardiac insufficiency. It is concluded that titin mutations have significant functional consequences and need to be studied intensively in the future.

The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton.

Ahnak is a ubiquitously expressed giant protein of 5643 amino acids implicated in cell differentiation and signal transduction. In a recent study, we demonstrated the association of ahnak with the regulatory beta2 subunit of the cardiac L-type Ca2+ channel. Here we identify the most carboxyl-terminal ahnak region (aa 5262-5643) to interact with recombinant beta2a as well as with beta2 and beta1a isoforms of native muscle Ca2+ channels using a panel of GST fusion proteins. Equilibrium sedimentation analysis revealed Kd values of 55 +/- 11 nM and 328 +/- 24 nM for carboxyl-terminal (aa 195-606) and amino-terminal (aa 1-200) truncates of the beta2a subunit, respectively. The same carboxyl-terminal ahnak region (aa 5262-5643) bound to G-actin and cosedimented with F-actin. Confocal microscopy of human left ventricular tissue localized the carboxyl-terminal ahnak portion to the sarcolemma including the T-tubular system and the intercalated disks of cardiomyocytes. These results suggest that ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin-based cytoskeleton to the L-type Ca2+ channel.

The carboxyl-terminal ahnak domain induces actin bundling and stabilizes muscle contraction.

Ahnak, a 700 kDa protein, is expressed in a variety of cells and has been implicated in different cell-type-specific functions. In the human heart, we observed an endogenous carboxyl-terminal 72 kDa ahnak fragment that copurified with myofibrillar proteins. Immunocytochemistry combined with confocal microscopy localized this fragment to the intercalated discs and close to the Z-line of cardiomyocytes. No endogenous myofibrillar ahnak fragment was observed in the skeletal muscle. We elucidated the role of the recombinant carboxyl-terminal ahnak fragment (ahnak-C2) in actin filament organization and in the function of muscle fibers. Addition of ahnak-C2 to actin filaments induced filament bundling into paracrystalline-like structures as revealed by electron microscopy. Incubation of demembranated skeletal muscle fibers with ahnak-C2 attenuated the decline in isometric force development upon repeated contraction-relaxation cycles. Our results suggest that the carboxyl-terminal ahnak domain exerts a stabilizing effect on muscle contractility via its interaction with actin of thin filaments.

Carvedilol improves myocardial contractility compared with metoprolol in patients with chronic hibernating myocardium after revascularization.

BACKGROUND: We tested the hypothesis of whether carvedilol delays morphologic degeneration and improves functional outcome compared with metoprolol tartrate in patients with hibernating myocardium undergoing surgical revascularization. We have previously shown that patients with chronic hibernating myocardium undergo progressive cellular degeneration and fibrosis. METHODS: Twenty patients with multivessel coronary artery disease revascularization and hibernating myocardium as assessed by technetium-99m perfusion scintigraphy and fluorine-18-fluorodeoxyglucose positron emission tomography were randomized to receive either carvedilol or metoprolol tartrate for at least 2 months before surgery, and this was continued for 7 months postoperatively. Left ventricular ejection fraction and regional wall motion abnormalities were assessed by left ventriculography at baseline and 7 months postoperatively. Intraoperative transmural needle biopsy samples were obtained for microscopic analysis. RESULTS: Postoperatively, the ejection fraction increased from 31% +/- 5% to 44% +/- 4% (P < .005) in the carvedilol group (n = 10), and from 30% +/- 6% to 40% +/- 6% in the metoprolol tartrate group (P < .05 vs preoperatively and vs carvedilol). Wall motion abnormalities in the carvedilol group improved from -2.1 +/- 0.4 to -0.6 +/- 0.5 (P < .05) and from -2.3 +/- 0.5 to -1.6 +/- 0.6 in the metoprolol tartrate group (P < .05 vs preoperatively and vs carvedilol). Microscopic analysis after 72 +/- 18 days of either treatment showed mild cardiomyocyte degeneration and moderate-to-severe fibrosis (28% +/- 7%) in the carvedilol group compared with moderate cardiomyocyte degeneration and moderate-to-severe fibrosis (33% +/- 6%) in the metoprolol tartrate group. Apoptosis, as assessed by the terminal deoxynucleotidyl transferase nick end labeling method, was observed in only 1 patient in each group. CONCLUSIONS: Carvedilol treatment of hibernating myocardium results in improved functional recovery after revascularization compared with metoprolol tartrate, and this might partially be related to reduced cardiomyocyte degeneration.

Connexin 43 expression and distribution in compensated and decompensated cardiac hypertrophy in patients with aortic stenosis.

OBJECTIVES: Gap junctions (GJ) are important determinants of conduction. In advanced heart failure alterations of the major ventricular GJ protein, connexin 43 (Cx43) are found. However, changes in Cx43 expression during the progression from compensated cardiac hypertrophy to heart failure, especially in humans, have not been studied extensively. The aim of the present study was to investigate changes in Cx43 expression and distribution in compensated and decompensated left ventricular (LV) hypertrophy in pressure-overloaded human hearts with valvular aortic stenosis (AS). METHODS: We measured Cx43 levels by Western blot and quantitative immunoconfocal microscopy of LV septum biopsies from three groups of patients with AS (group I (n=9): ejection fraction (EF)>50%; group II (n=12): EF 30-50%; group III (n=9): EF<30%). LV biopsies from six patients with mitral valve stenosis and two donor hearts served as controls. RESULTS: Only the early phase of LV hypertrophy (AS-I) was characterized by extensive Cx43 lateral staining. As compared to controls, the AS-I group showed a 44.3% increase in Cx43 protein, which was reflected in an augmented number of GJs per 100 microm(2) intercalated disc area (control: 62.5+/-6.4 vs. AS-I: 79.8+/-4, p<0.001) and an increased GJ surface density (control: 0.00547 vs. AS-I: 0.00724 microm(2)/microm(3), p<0.01). Decompensated LV hypertrophy (AS-III) was specified by reduced percentage of the Cx43 signal per myocyte area (control: 1.74% vs. AS-III: 1.31%, p<0.01) or per intercalated disc (control: 18.3% vs. AS-III: 11.3%, p<0.005). Mean GJ area and GJ number per intercalated discs in the AS-III group were decreased significantly by, respectively, 42.5% and 36.4% as compared to control. In addition, decompensated LV myocardium showed a markedly heterogeneous spatial distribution of Cx43. CONCLUSION: The quantity and spatial distribution of Cx43 differs markedly between compensated and decompensated LV hypertrophy in human patients with AS. Upregulation of Cx43 in compensated hypertrophy may represent the immediate adaptive response to increased load, whereas diminished and heterogeneous Cx43 distribution in decompensated hypertrophy may play maladaptive roles culminating in heart failure and ventricular arrhythmias.