Michael Sacherer

Biomechanical properties and microstructure of human ventricular myocardium

UNLABELLED In the multidisciplinary field of heart research it is of utmost importance to identify accurate myocardium material properties for the description of phenomena such as mechano-electric feedback or heart wall thickening. A rationally-based material model is required to understand the highly nonlinear mechanics of complex structures such as the passive myocardium under different loading conditions. Unfortunately, to date there are no experimental data of human heart tissues available to estimate material parameters and to develop adequate material models. This study aimed to determine biaxial extension and triaxial shear properties and the underlying microstructure of the passive human ventricular myocardium. Using new state-of-the-art equipment, planar biaxial extension tests were performed to determine the biaxial extension properties of the passive ventricular human myocardium. Shear properties of the myocardium were examined by triaxial simple shear tests performed on small cubic specimens excised from an adjacent region of the biaxial extension specimens. The three-dimensional microstructure was investigated through second-harmonic generation (SHG) microscopy on optically cleared tissues, which emphasized the 3D orientation and dispersion of the myofibers and adjacent collagen fabrics. The results suggest that the passive human LV myocardium under quasi-static and dynamic multiaxial loadings is a nonlinear, anisotropic (orthotropic), viscoelastic and history-dependent soft biological material undergoing large deformations. Material properties of the tissue components along local microstructural axes drive the nonlinear and orthotropic features of the myocardium. SHG microscopy investigation revealed detailed information about the myocardial microstructure due to its high resolution. It enabled the identification of structural parameters such as the fiber and the sheet orientations and corresponding dispersions. With this complete set of material data, a sophisticated material model and associated material parameters can be defined for a better description of the biomechanical response of the ventricular myocardium in humans. Such a model will lead to more accurate computational simulations to better understand the fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases. STATEMENT OF SIGNIFICANCE Unfortunately, to date there are no experimental data of human heart tissues available for material parameter estimation and the development of adequate material models. In this manuscript novel biaxial tensile and shear test data at different specimen orientations are presented, which allowed to adequately capture the direction-dependent material response. With these complete sets of mechanical data, combined with their underlying microstructural data (also presented herein), sophisticated material models and associated material parameters can be defined for the description of the mechanical behavior of the ventricular myocardium in humans. Such models will lead to accurate computational simulations to better understand the fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases.

JTV519 (K201) reduces sarcoplasmic reticulum Ca2+ leak and improves diastolic function in vitro in murine and human non-failing myocardium

BACKGROUND AND PURPOSE Ca2+ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca2+ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4‐benzothiazepine derivative and multi‐channel blocker, stabilizes RyR2s and decrease SR Ca2+ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non‐failing human myocardium and explored underlying mechanisms.

Vascular Bioactivation of Nitroglycerin Is Catalyzed by Cytosolic Aldehyde Dehydrogenase-2

Rationale: According to general view, aldehyde dehydrogenase-2 (ALDH2) catalyzes the high-affinity pathway of vascular nitroglycerin (GTN) bioactivation in smooth muscle mitochondria. Despite having wide implications to GTN pharmacology and raising many questions that are still unresolved, mitochondrial bioactivation of GTN in blood vessels is still lacking experimental support. Objective: In the present study, we investigated whether bioactivation of GTN is affected by the subcellular localization of ALDH2 using immortalized ALDH2-deficient aortic smooth muscle cells and mouse aortas with selective overexpression of the enzyme in either cytosol or mitochondria. Methods and Results: Quantitative Western blotting revealed that ALDH2 is mainly cytosolic in mouse aorta and human coronary arteries, with only approximately 15% (mouse) and approximately 5% (human) of the enzyme being localized in mitochondria. Infection of ALDH2-deficient aortic smooth muscle cells or isolated aortas with adenovirus containing ALDH2 cDNA with or without the mitochondrial signal peptide sequence led to selective expression of the protein in mitochondria and cytosol, respectively. Cytosolic overexpression of ALDH2 restored GTN-induced relaxation and GTN denitration to wild-type levels, whereas overexpression in mitochondria (6-fold vs wild-type) had no effect on relaxation. Overexpression of ALDH2 in the cytosol of ALDH2-deficient aortic smooth muscle cells led to a significant increase in GTN denitration and cyclic GMP accumulation, whereas mitochondrial overexpression had no effect. Conclusions: The data indicate that vascular bioactivation of GTN is catalyzed by cytosolic ALDH2. Mitochondrial GTN metabolism may contribute to oxidative stress-related adverse effects of nitrate therapy and the development of nitrate tolerance.

Intracellular Dyssynchrony of Diastolic Cytosolic [Ca2+] Decay in Ventricular Cardiomyocytes in Cardiac Remodeling and Human Heart Failure

Rationale: Synchronized release of Ca2+ into the cytosol during each cardiac cycle determines cardiomyocyte contraction. Objective: We investigated synchrony of cytosolic [Ca2+] decay during diastole and the impact of cardiac remodeling. Methods and Results: Local cytosolic [Ca2+] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca2+] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca2+ release. Stimulation of sarcoplasmic reticulum Ca2+ ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca2+] decay (TAUglobal). Na+/Ca2+ exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca2+ uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. Conclusions: In cardiomyocytes, cytosolic [Ca2+] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca2+] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

Early Remodeling of Perinuclear Ca2+ Stores and Nucleoplasmic Ca2+ Signaling During the Development of Hypertrophy and Heart Failure

Background— A hallmark of heart failure is impaired cytoplasmic Ca2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca2+ handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. Methods and Results— Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca2+ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca2+ transporters. These changes were associated with altered nucleoplasmic Ca2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca2+ levels with diminished and prolonged nuclear Ca2+ transients and slowed intranuclear Ca2+ diffusion. Altered nucleoplasmic Ca2+ levels were translated to higher activation of nuclear Ca2+/calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca2+ changes that are typical of heart failure. Conclusions— During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca2+ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling.

Quantification of Shear Deformations and Corresponding Stresses in the Biaxially Tested Human Myocardium

One goal of cardiac research is to perform numerical simulations to describe/reproduce the mechanoelectrical function of the human myocardium in health and disease. Such simulations are based on a complex combination of mathematical models describing the passive mechanical behavior of the myocardium and its electrophysiology, i.e., the activation of cardiac muscle cells. The problem in developing adequate constitutive models is the shortage of experimental data suitable for detailed parameter estimation in specific functional forms. A combination of shear and biaxial extension tests with different loading protocols on different specimen orientations is necessary to capture adequately the direction-dependent (orthotropic) response of the myocardium. In most experimental animal studies, where planar biaxial extension tests on the myocardium have been conducted, the generated shear stresses were neither considered nor discussed. Hence, in this study a method is presented which allows the quantification of shear deformations and related stresses. It demonstrates an approach for experimenters as to how the generation of these shear stresses can be minimized during mechanical testing. Experimental results on 14 passive human myocardial specimens, obtained from nine human hearts, show the efficiency of this newly developed method. Moreover, the influence of the clamping technique of the specimen, i.e., the load transmission between the testing device and the tissue, on the stress response is determined by testing an isotropic material (Latex). We identified that the force transmission between the testing device and the specimen by means of hooks and cords does not influence the performed experiments. We further showed that in-plane shear stresses definitely exist in biaxially tested human ventricular myocardium, but can be reduced to a minimum by preparing the specimens in an appropriate manner. Moreover, we showed whether shear stresses can be neglected when performing planar biaxial extension tests on fiber-reinforced materials. The used method appears to be robust to quantify normal and shear deformations and corresponding stresses in biaxially tested human myocardium. This method can be applied for the mechanical characterization of any fiber-reinforced material using planar biaxial extension tests.

Involvement Of Vascular Aldosterone Synthase In Phosphate-Induced Osteogenic Transformation Of Vascular Smooth Muscle Cells

Vascular calcification resulting from hyperphosphatemia is a major determinant of mortality in chronic kidney disease (CKD). Vascular calcification is driven by aldosterone-sensitive osteogenic transformation of vascular smooth muscle cells (VSMCs). We show that even in absence of exogenous aldosterone, silencing and pharmacological inhibition (spironolactone, eplerenone) of the mineralocorticoid receptor (MR) ameliorated phosphate-induced osteo-/chondrogenic transformation of primary human aortic smooth muscle cells (HAoSMCs). High phosphate concentrations up-regulated aldosterone synthase (CYP11B2) expression in HAoSMCs. Silencing and deficiency of CYP11B2 in VSMCs ameliorated phosphate-induced osteogenic reprogramming and calcification. Phosphate treatment was followed by nuclear export of APEX1, a CYP11B2 transcriptional repressor. APEX1 silencing up-regulated CYP11B2 expression and stimulated osteo-/chondrogenic transformation. APEX1 overexpression blunted the phosphate-induced osteo-/chondrogenic transformation and calcification of HAoSMCs. Cyp11b2 expression was higher in aortic tissue of hyperphosphatemic klotho-hypomorphic (kl/kl) mice than in wild-type mice. In adrenalectomized kl/kl mice, spironolactone treatment still significantly ameliorated aortic osteoinductive reprogramming. Our findings suggest that VSMCs express aldosterone synthase, which is up-regulated by phosphate-induced disruption of APEX1-dependent gene suppression. Vascular CYP11B2 may contribute to stimulation of VSMCs osteo-/chondrogenic transformation during hyperphosphatemia.

SGK1 induces vascular smooth muscle cell calcification through NF-κB signaling

&NA; Medial vascular calcification, associated with enhanced mortality in chronic kidney disease (CKD), is fostered by osteo‐/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Here, we describe that serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) was upregulated in VSMCs under calcifying conditions. In primary human aortic VSMCs, overexpression of constitutively active SGK1S422D, but not inactive SGK1K127N, upregulated osteo‐/chondrogenic marker expression and activity, effects pointing to increased osteo‐/chondrogenic transdifferentiation. SGK1S422D induced nuclear translocation and increased transcriptional activity of NF‐&kgr;B. Silencing or pharmacological inhibition of IKK abrogated the osteoinductive effects of SGK1S422D. Genetic deficiency, silencing, and pharmacological inhibition of SGK1 dissipated phosphate‐induced calcification and osteo‐/chondrogenic transdifferentiation of VSMCs. Aortic calcification, stiffness, and osteo‐/chondrogenic transdifferentiation in mice following cholecalciferol overload were strongly reduced by genetic knockout or pharmacological inhibition of Sgk1 by EMD638683. Similarly, Sgk1 deficiency blunted vascular calcification in apolipoprotein E‐deficient mice after subtotal nephrectomy. Treatment of human aortic smooth muscle cells with serum from uremic patients induced osteo‐/chondrogenic transdifferentiation, effects ameliorated by EMD638683. These observations identified SGK1 as a key regulator of vascular calcification. SGK1 promoted vascular calcification, at least partly, via NF‐&kgr;B activation. Inhibition of SGK1 may, thus, reduce the burden of vascular calcification in CKD.

Early Remodelling of Perinuclear Ca2+ Stores andNucleoplasmic Ca2+ Signalling During the Development of Hypertrophyand Heart Failure

Background— A hallmark of heart failure is impaired cytoplasmic Ca2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca2+ handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. Methods and Results— Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca2+ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca2+ transporters. These changes were associated with altered nucleoplasmic Ca2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca2+ levels with diminished and prolonged nuclear Ca2+ transients and slowed intranuclear Ca2+ diffusion. Altered nucleoplasmic Ca2+ levels were translated to higher activation of nuclear Ca2+/calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca2+ changes that are typical of heart failure. Conclusions— During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca2+ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling.

Early Remodeling of Perinuclear Ca2+ Stores and Nucleoplasmic Ca2+ Signaling During the Development of Hypertrophy and Heart FailureCLINICAL PERSPECTIVE

Background— A hallmark of heart failure is impaired cytoplasmic Ca2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca2+ handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. Methods and Results— Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca2+ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca2+ transporters. These changes were associated with altered nucleoplasmic Ca2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca2+ levels with diminished and prolonged nuclear Ca2+ transients and slowed intranuclear Ca2+ diffusion. Altered nucleoplasmic Ca2+ levels were translated to higher activation of nuclear Ca2+/calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca2+ changes that are typical of heart failure. Conclusions— During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca2+ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling.