Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy

Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, α-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin ↔ Glu-tubulin) and then irreversible deglutamination (Glu-tubulin → Δ2-tubulin), such that Glu- and Δ2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Δ2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Δ2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Δ2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.     

Biventricular Remodeling in Murine Models of Right Ventricular Pressure Overload

Right ventricular (RV) failure is a major cause of mortality in acute or chronic lung disease and left heart failure. The objective of this study was to demonstrate a percutaneous approach to study biventricular hemodynamics in murine models of primary and secondary RV pressure overload (RVPO) and further explore biventricular expression of two key proteins that regulate cardiac remodeling: calcineurin and transforming growth factor beta 1 (TGFβ1).

Methods

Adult, male mice underwent constriction of the pulmonary artery or thoracic aorta as models of primary and secondary RVPO, respectively. Conductance catheterization was performed followed by tissue analysis for changes in myocyte hypertrophy and fibrosis.

Results

Both primary and secondary RVPO decreased biventricular stroke work however RV instantaneous peak pressure (dP/dt_max) and end-systolic elastance (Ees) were preserved in both groups compared to controls. In contrast, left ventricular (LV) dP/dt_max and LV-Ees were unchanged by primary, but reduced in the secondary RVPO group. The ratio of RV:LV ventriculo-arterial coupling was increased in primary and reduced in secondary RVPO. Primary and secondary RVPO increased RV mass, while LV mass decreased in primary and increased in the secondary RVPO groups. RV fibrosis and hypertrophy were increased in both groups, while LV fibrosis and hypertrophy were increased in secondary RVPO only. RV calcineurin expression was increased in both groups, while LV expression increased in secondary RVPO only. Biventricular TGFβ1 expression was increased in both groups.

Conclusion

These data identify distinct effects of primary and secondary RVPO on biventricular structure, function, and expression of key remodeling pathways.     

Cardiac Hypertrophy Involves Both Myocyte Hypertrophy and Hyperplasia in Anemic Zebrafish

Background

An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart.

Methodology/Principal Findings

Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ).

Conclusions/Significance

The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized.     

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50–60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.     

Connective Tissue Growth Factor Overexpression in Cardiomyocytes Promotes Cardiac Hypertrophy and Protection against Pressure Overload

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca²⁺ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF''s profibrotic function in the heart.     

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.     

Temporal and Molecular Analyses of Cardiac Extracellular Matrix Remodeling following Pressure Overload in Adiponectin Deficient Mice

Adiponectin, circulating levels of which are reduced in obesity and diabetes, mediates cardiac extracellular matrix (ECM) remodeling in response to pressure overload (PO). Here, we performed a detailed temporal analysis of progressive cardiac ECM remodelling in adiponectin knockout (AdKO) and wild-type (WT) mice at 3 days and 1, 2, 3 and 4 weeks following the induction of mild PO via minimally invasive transverse aortic banding. We first observed that myocardial adiponectin gene expression was reduced after 4 weeks of PO, whereas increased adiponectin levels were detected in cardiac homogenates at this time despite decreased circulating levels of adiponectin. Scanning electron microscopy and Masson’s trichrome staining showed collagen accumulation increased in response to 2 and 4 weeks of PO in WT mice, while fibrosis in AdKO mice was notably absent after 2 weeks but highly apparent after 4 weeks of PO. Time and intensity of fibroblast appearance after PO was not significantly different between AdKO and WT animals. Gene array analysis indicated that MMP2, TIMP2, collagen 1α1 and collagen 1α3 were induced after 2 weeks of PO in WT but not AdKO mice. After 4 weeks MMP8 was induced in both genotypes, MMP9 only in WT mice and MMP1α only in AdKO mice. Direct stimulation of primary cardiac fibroblasts with adiponectin induced a transient increase in total collagen detected by picrosirius red staining and collagen III levels synthesis, as well as enhanced MMP2 activity detected via gelatin zymography. Adiponectin also enhanced fibroblast migration and attenuated angiotensin-II induced differentiation to a myofibroblast phenotype. In conclusion, these data indicate that increased myocardial bioavailability of adiponectin mediates ECM remodeling following PO and that adiponectin deficiency delays these effects.     

Signaling to Cardiac Hypertrophy: Insights from Human and Mouse RASopathies

Cardiac hypertrophy is the heart’s response to a variety of extrinsic and intrinsic stimuli, some of which might finally lead up to a maladaptive state. An integral part of the pathogenesis of the hypertrophic cardiomyopathy disease (HCM) is the activation of the rat sarcoma (RAS)/RAF/MEK (mitogen-activated protein kinase kinase)/MAPK (mitogen-activated protein kinase) cascade. Therefore, the molecular signaling involving RAS has been the subject of intense research efforts, particularly after the identification of the RASopathies. These constitute a class of developmental disorders caused by germline mutations affecting proteins contributing to the RAS pathway. Among other phenotypic features, a subset of these syndromes is characterized by HCM, prompting researchers and clinicians to delve into the chief signaling constituents of cardiac hypertrophy. In this review, we summarize current advances in the knowledge of the molecular signaling events involved in the pathogenesis of cardiac hypertrophy through work completed on patients and on genetically manipulated animals with HCM and RASopathies. Important insights are drawn from the recognition of parallels between cardiac hypertrophy and cancer. Future research promises to further elucidate the complex molecular interactions responsible for cardiac hypertrophy, possibly pointing the way for the identification of new specific targets for the treatment of HCM.     

Signaling to Cardiac Hypertrophy: Insights from Human and Mouse RASopathies

Cardiac hypertrophy is the heart's response to a variety of extrinsic and intrinsic stimuli, some of which might finally lead up to a maladaptive state. An integral part of the pathogenesis of the hypertrophic cardiomyopathy disease (HCM) is the activation of the rat sarcoma (RAS)/RAF/MEK (mitogen-activated protein kinase kinase)/MAPK (mitogen-activated protein kinase) cascade. Therefore, the molecular signaling involving RAS has been the subject of intense research efforts, particularly after the identification of the RASopathies. These constitute a class of developmental disorders caused by germline mutations affecting proteins contributing to the RAS pathway. Among other phenotypic features, a subset of these syndromes is characterized by HCM, prompting researchers and clinicians to delve into the chief signaling constituents of cardiac hypertrophy. In this review, we summarize current advances in the knowledge of the molecular signaling events involved in the pathogenesis of cardiac hypertrophy through work completed on patients and on genetically manipulated animals with HCM and RASopathies. Important insights are drawn from the recognition of parallels between cardiac hypertrophy and cancer. Future research promises to further elucidate the complex molecular interactions responsible for cardiac hypertrophy, possibly pointing the way for the identification of new specific targets for the treatment of HCM.     

Spatial Repolarization Heterogeneity Detected by Magnetocardiography Correlates with Cardiac Iron Overload and Adverse Cardiac Events in Beta-Thalassemia Major

Background

Patients with transfusion-dependent beta-thalassemia major (TM) are at risk for myocardial iron overload and cardiac complications. Spatial repolarization heterogeneity is known to be elevated in patients with certain cardiac diseases, but little is known in TM patients. The purpose of this study was to evaluate spatial repolarization heterogeneity in patients with TM, and to investigate the relationships between spatial repolarization heterogeneity, cardiac iron load, and adverse cardiac events.

Methods and Results

Fifty patients with TM and 55 control subjects received 64-channel magnetocardiography (MCG) to determine spatial repolarization heterogeneity, which was evaluated by a smoothness index of QT_c (SI-QT_c), a standard deviation of QT_c (SD-QT_c), and a QT_c dispersion. Left ventricular function and myocardial T2* values were assessed by cardiac magnetic resonance. Patients with TM had significantly greater SI-QT_c, SD-QT_c, and QT_c dispersion compared to the control subjects (all p values<0.001). Spatial repolarization heterogeneity was even more pronounced in patients with significant iron overload (T2*<20 ms, n = 20) compared to those with normal T2* (all p values<0.001). Log_e cardiac T2* correlated with SI-QT_c (r = −0.609, p<0.001), SD-QT_c (r = −0.572, p<0.001), and QT_c dispersion (r = −0.622, p<0.001), while all these indices had no relationship with measurements of the left ventricular geometry or function. At the time of study, 10 patients had either heart failure or arrhythmia. All 3 indices of repolarization heterogeneity were related to the presence of adverse cardiac events, with areas under the receiver operating characteristic curves (ranged between 0.79 and 0.86), similar to that of cardiac T2*.

Conclusions

Multichannel MCG demonstrated that patients with TM had increased spatial repolarization heterogeneity, which is related to myocardial iron load and adverse cardiac events.     

Multiple Residues Contribute to the Inability of Murine CCR-5 To Function as a Coreceptor for Macrophage-Tropic Human Immunodeficiency Virus Type 1 Isolates

Infection of CD4-positive cells by human immunodeficiency virus type 1 (HIV-1) requires functional interaction of the viral envelope protein with a coreceptor belonging to the chemokine receptor family of seven-membrane-spanning receptors. For the majority of macrophage-tropic HIV-1 isolates, the physiologically relevant coreceptor is the human CCR-5 (hCCR-5) receptor. Although the murine homolog of CCR-5 (mCCR-5) is unable to mediate HIV-1 infection, chimeric hCCR-5/mCCR-5 molecules containing single extracellular domains derived from hCCR-5 are effective coreceptors for certain macrophage-tropic HIV-1 isolates. Here, we have sought to identify residues in hCCR-5 critical for HIV-1 infection by substitution of mCCR-5-derived residues into the context of functional chimeric hCCR-5/mCCR-5 receptor molecules. Using this strategy, we demonstrate that residues 7, 13, and 15 in the first extracellular domain and residue 180 in the third extracellular domain of CCR-5 are important for HIV-1 envelope-mediated membrane fusion. Of interest, certain substitutions, for example, at residues 184 and 185 in the third extracellular domain, have no phenotype when introduced individually but strongly inhibit hCCR-5 coreceptor function when present together. We hypothesize that these changes, which do not preclude chemokine receptor function, may inhibit a conformational transition in hCCR-5 that contributes to HIV-1 infection. Finally, we report that substitution of glycine for valine at residue 5 in CCR-5 can significantly enhance the level of envelope-dependent cell fusion by expressing cells. The diversity of the mutant phenotypes observed in this mutational analysis, combined with their wide distribution across the extracellular regions of CCR-5, emphasizes the complexity of the interaction between HIV-1 envelope and coreceptor.Infection of cells by human immunodeficiency virus type 1 (HIV-1) requires interaction of the viral envelope protein with not only CD4 but also a second cell surface molecule, termed a coreceptor (reviewed in reference 19). Coreceptor usage varies significantly among different HIV-1 isolates, although all known coreceptors are members of the G-protein-coupled chemokine receptor family of seven-membrane-spanning receptors. The primary coreceptor used by non-syncytium-inducing, macrophage-tropic (M-tropic) HIV-1 isolates, which constitute the majority of primary isolates, is CCR-5 (1, 6, 8, 12, 27). In contrast, syncytium-inducing, T-cell-line-adapted (T-tropic) HIV-1 isolates predominantly use CXCR-4 as a coreceptor (13). Other chemokine receptors utilized by a small percentage of generally dualtropic HIV-1 isolates include CCR-2b and CCR-3 (6, 11). The importance of two orphan chemokine receptors, termed Bonzo/STRL33 and BOB/GPR15, in infection by HIV-1 remains to be established, although these proteins were recently shown to serve as coreceptors for several simian immunodeficiency virus and HIV-2 isolates (2, 9). The critical importance of CCR-5 for infection by primary, M-tropic HIV-1 isolates, however, has been highlighted by the finding that a small percentage of humans lack a functional CCR-5 gene and as a result appear highly, although not completely, resistant to infection by HIV-1 (17, 22). Importantly, primary T cells derived from such individuals are refractory to infection by M-tropic HIV-1 isolates in vitro (17, 22, 27), thus demonstrating that CCR-5 is the physiologically relevant coreceptor for the majority of primary isolates.At present, relatively little is known about how the viral envelope and coreceptor interact, although it appears clear that interaction is dependent upon a prior conformational shift induced by binding of the envelope gp120 subunit to CD4 (24, 26). This in turn is believed to lead to the formation of a ternary complex, consisting of gp120, coreceptor, and CD4, on the surface of the target cell (15, 24, 26). It is unknown how this protein complex then induces the fusion of the viral and host cell membranes, although the envelope gp41 subunit is believed to play a critical role at this stage.An important unresolved question is the identity of the amino acid residues in gp120 and the coreceptor that interact during infection. However, it is well established that HIV-1 tropism, and hence coreceptor usage, is largely controlled by a small number of residues located in the envelope V3 loop (6, 14, 23, 25). Efforts to identify residues in the CCR-5 coreceptor involved in mediating infection have thus far largely focused on the functional analysis of chimeric receptors generated with human CCR-5 (hCCR-5) and a chemokine receptor lacking coreceptor function, such as the murine CCR-5 homolog (mCCR-5) (3, 5, 20, 21). These studies have led to three major conclusions. Firstly, the residues in hCCR-5 involved in mediating HIV-1 infection are diffuse, being located on at least three of the four extracellular domains of CCR-5. Secondly, these residues are functionally redundant, so that several distinct regions of hCCR-5 can suffice independently to confer coreceptor function when substituted into mCCR-5. Lastly, different HIV-1 envelope proteins interact differently with CCR-5, such that CCR-5 residues important for mediating fusion by one envelope protein may be largely irrelevant to the interaction of CCR-5 with a second envelope protein. Overall, these data demonstrate that the envelope–CCR-5 interaction is likely to be highly complex and to involve the interaction of multiple residues in both proteins.As noted above, the mCCR-5 chemokine receptor, despite extensive sequence similarity to hCCR-5, fails to function as an HIV-1 coreceptor (3, 5, 20). Therefore, it is apparent that one or more of the 20 extracellular residues that differ between mCCR-5 and hCCR-5 must contribute to the interaction with the HIV-1 envelope protein. Using mutational analysis in the context of chimeric mCCR-5/hCCR-5 receptors, we have now identified several residues, located in three of the four extracellular domains of hCCR-5, that play roles in mediating infection by HIV-1.     

Cardiac Specific Expression of Threonine 5 to Alanine Mutant Sarcolipin Results in Structural Remodeling and Diastolic Dysfunction

The functional importance of threonine 5 (T5) in modulating the activity of sarcolipin (SLN), a key regulator of sarco/endoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) pump was studied using a transgenic mouse model with cardiac specific expression of threonine 5 to alanine mutant SLN (SLNT5A). In these transgenic mice, the SLNT5A protein replaces the endogenous SLN in atria, while maintaining the total SLN content. The cardiac specific expression of SLNT5A results in severe cardiac structural remodeling accompanied by bi-atrial enlargement. Biochemical analyses reveal a selective downregulation of SR Ca²⁺ handling proteins and a reduced SR Ca²⁺ uptake both in atria and in the ventricles. Optical mapping analysis shows slower action potential propagation in the transgenic mice atria. Doppler echocardiography and hemodynamic measurements demonstrate a reduced atrial contractility and an impaired diastolic function. Together, these findings suggest that threonine 5 plays an important role in modulating SLN function in the heart. Furthermore, our studies suggest that alteration in SLN function can cause abnormal Ca²⁺ handling and subsequent cardiac remodeling and dysfunction.     

Nocturnal Blood Pressure Pattern Affects Left Ventricular Remodeling and Late Gadolinium Enhancement in Patients with Hypertension and Left Ventricular Hypertrophy

Background

Left ventricular hypertrophy (LVH) is an independent predictor of cardiac mortality, regardless of its etiology. Previous studies have shown that high nocturnal blood pressure (BP) affects LV geometry in hypertensive patients. It has been suggested that continuous pressure overload affects the development of LVH, but it is unknown whether persistent pressure influences myocardial fibrosis or whether the etiology of LVH is associated with myocardial fibrosis. Comprehensive cardiac magnetic resonance (CMR) including the late gadolinium enhancement (LGE) technique can evaluate both the severity of changes in LV geometry and myocardial fibrosis. We tested the hypothesis that the nocturnal non-dipper BP pattern causes LV remodeling and fibrosis in patients with hypertension and LVH.

Methods

Forty-seven hypertensive patients with LVH evaluated by echocardiography (29 men, age 73.0±10.4 years) were examined by comprehensive CMR and 24-h ambulatory blood pressure monitoring (ABPM).

Results and Conclusions

Among the 47 patients, twenty-four had nocturnal non-dipper BP patterns. Patients with nocturnal non-dipper BP patterns had larger LV masses and scar volumes independent of etiologies than those in patients with dipper BP patterns (p = 0.035 and p = 0.015, respectively). There was no significant difference in mean 24-h systolic BP between patients with and without nocturnal dipper BP patterns (p = 0.367). Among hypertensive patients with LVH, the nocturnal non-dipper blood pressure pattern is associated with both LV remodeling and myocardial fibrosis independent of LVH etiology.