Markus Wallner

Mild hypothermia attenuates circulatory and pulmonary dysfunction during experimental endotoxemia.

Objective:We tested whether mild hypothermia impacts on circulatory and respiratory dysfunction during experimental endotoxemia. Design:Randomized controlled prospective experimental study. Setting:Large animal facility, Medical University of Graz, Austria. Subjects:Thirteen anesthetized and mechanically ventilated pigs. Interventions:Lipopolysaccharide was administered for 4 hours. With the beginning of lipopolysaccharide infusion, animals were assigned to either normothermia (38°C, n = 7) or mild hypothermia (33°C, n = 6, intravascular cooling) and followed for 8 hours in total. Measurements and Main Results:At the end of the protocol, cardiac output was lower in mild hypothermia than in normothermia (4.5 ± 0.4 L/min vs 6.6 ± 0.4 L/min, p < 0.05), but systemic vascular resistance (885 ± 77 dyn·s/cm5 vs 531 ± 29 dyn·s/cm5, p < 0.05) and (77% ± 6% vs 54% ± 3%, p < 0.05) were higher. Indices of left ventricular contractility in vivo were not different between groups. The high-frequency band in spectral analysis of heart rate variability indicated a better preserved vagal autonomic modulation of sinuatrial node activity in mild hypothermia versus normothermia (87 ± 5 vs 47 ± 5, normalized units, p < 0.05). Plasma norepinephrine levels were elevated compared with baseline in normothermia (2.13 ± 0.27 log pg/mL vs 0.27 ± 0.17 log pg/mL, p < 0.05) but not in mild hypothermia (1.02 ± 0.31 vs 0.55 ± 0.26, p = not significant). At 38°C in vitro, left ventricular muscle strips isolated from the mild hypothermia group had a higher force response to isoproterenol. SaO2 (100% ± 0% vs 92% ± 3%, p < 0.05) and the oxygenation index (PO2/FIO2, 386 ± 52 mm Hg vs 132 ± 32 mm Hg, p < 0.05) were substantially higher in mild hypothermia versus normothermia. Plasma cytokine levels were not consistently different between groups (interleukin 10) or higher (tumor necrosis factor-&agr; and interleukin 6 and 8) during mild hypothermia versus normothermia. Conclusion:The induction of mild hypothermia attenuates cardiac and respiratory dysfunction and counteracts sympathetic activation during experimental endotoxemia. This was not associated with lower plasma cytokine levels, indicating a reduction of cytokine responsiveness by mild hypothermia.

Role of STIM1 (Stromal Interaction Molecule 1) in Hypertrophy-Related Contractile Dysfunction

Rationale: Pathological increases in cardiac afterload result in myocyte hypertrophy with changes in myocyte electrical and mechanical phenotype. Remodeling of contractile and signaling Ca2+ occurs in pathological hypertrophy and is central to myocyte remodeling. STIM1 (stromal interaction molecule 1) regulates Ca2+ signaling in many cell types by sensing low endoplasmic reticular Ca2+ levels and then coupling to plasma membrane Orai channels to induce a Ca2+ influx pathway. Previous reports suggest that STIM1 may play a role in cardiac hypertrophy, but its role in electrical and mechanical phenotypic alterations is not well understood. Objective: To define the contributions of STIM1-mediated Ca2+ influx on electrical and mechanical properties of normal and diseased myocytes, and to determine whether Orai channels are obligatory partners for STIM1 in these processes using a clinically relevant large animal model of hypertrophy. Methods and Results: Cardiac hypertrophy was induced by slow progressive pressure overload in adult cats. Hypertrophied myocytes had increased STIM1 expression and activity, which correlated with altered Ca2+-handling and action potential (AP) prolongation. Exposure of hypertrophied myocytes to the Orai channel blocker BTP2 caused a reduction of AP duration and reduced diastolic Ca2+ spark rate. BTP2 had no effect on normal myocytes. Forced expression of STIM1 in cultured adult feline ventricular myocytes increased diastolic spark rate and prolonged AP duration. STIM1 expression produced an increase in the amount of Ca2+ stored within the sarcoplasmic reticulum and activated Ca2+/calmodulin-dependent protein kinase II. STIM1 expression also increased spark rates and induced spontaneous APs. STIM1 effects were eliminated by either BTP2 or by coexpression of a dominant negative Orai construct. Conclusions: STIM1 can associate with Orai in cardiac myocytes to produce a Ca2+ influx pathway that can prolong the AP duration and load the sarcoplasmic reticulum and likely contributes to the altered electromechanical properties of the hypertrophied heart.Rationale: Pathological increases in cardiac afterload result in myocyte hypertrophy with changes in myocyte electrical and mechanical phenotype. Remodeling of contractile and signaling Ca 2+ occurs in pathological hypertrophy and is central to myocyte remodeling. Stromal Interaction Molecule 1 (STIM1) regulates Ca 2+ signaling in many cell types by sensing low endoplasmic reticular Ca 2+ levels and then coupling to plasma membrane Orai channels to induce a Ca 2+ influx pathway. Previous reports suggest that STIM1 may play a role in cardiac hypertrophy but its role in electrical and mechanical phenotypic alterations are not well understood. Objective: To define the contributions of STIM1-mediated Ca 2+ influx on electrical and mechanical properties of normal and diseased myocytes, and to determine if Orai channels are obligatory partners for STIM1 in these processes using a clinically relevant large animal model of hypertrophy. Methods and Results: Cardiac hypertrophy was induced by slow progressive pressure overload in adult cats. Hypertrophied myocytes had increased STIM1 expression and activity, which correlated with altered Ca 2+ handling and action potential (AP) prolongation. Exposure of hypertrophied myocytes to the Orai channel blocker BTP2 caused in a reduction of AP duration and reduced diastolic Ca 2+ spark rate. BTP2 had no effect on normal myocytes. Forced expression of STIM1 in cultured adult feline ventricular myocytes increased diastolic spark rate and prolonged AP duration. STIM1 expression produced an increase in the amount of Ca 2+ stored within the sarcoplasmic reticulum and activated Ca 2+ /calmodulin-dependent protein kinase II. STIM1 expression also increased spark rates and induced spontaneous APs. STIM1 effects were eliminated by either BTP2 or by co-expression of a dominant negative Orai construct. Conclusions: STIM1 can associate with Orai in cardiac myocytes to produce a Ca 2+ influx pathway that can prolong the AP duration and load the SR and likely contributes to the altered electromechanical properties of the hypertrophied heart.

Role of STIM1 in Hypertrophy-Related Contractile Dysfunction

Rationale: Pathological increases in cardiac afterload result in myocyte hypertrophy with changes in myocyte electrical and mechanical phenotype. Remodeling of contractile and signaling Ca2+ occurs in pathological hypertrophy and is central to myocyte remodeling. STIM1 (stromal interaction molecule 1) regulates Ca2+ signaling in many cell types by sensing low endoplasmic reticular Ca2+ levels and then coupling to plasma membrane Orai channels to induce a Ca2+ influx pathway. Previous reports suggest that STIM1 may play a role in cardiac hypertrophy, but its role in electrical and mechanical phenotypic alterations is not well understood. Objective: To define the contributions of STIM1-mediated Ca2+ influx on electrical and mechanical properties of normal and diseased myocytes, and to determine whether Orai channels are obligatory partners for STIM1 in these processes using a clinically relevant large animal model of hypertrophy. Methods and Results: Cardiac hypertrophy was induced by slow progressive pressure overload in adult cats. Hypertrophied myocytes had increased STIM1 expression and activity, which correlated with altered Ca2+-handling and action potential (AP) prolongation. Exposure of hypertrophied myocytes to the Orai channel blocker BTP2 caused a reduction of AP duration and reduced diastolic Ca2+ spark rate. BTP2 had no effect on normal myocytes. Forced expression of STIM1 in cultured adult feline ventricular myocytes increased diastolic spark rate and prolonged AP duration. STIM1 expression produced an increase in the amount of Ca2+ stored within the sarcoplasmic reticulum and activated Ca2+/calmodulin-dependent protein kinase II. STIM1 expression also increased spark rates and induced spontaneous APs. STIM1 effects were eliminated by either BTP2 or by coexpression of a dominant negative Orai construct. Conclusions: STIM1 can associate with Orai in cardiac myocytes to produce a Ca2+ influx pathway that can prolong the AP duration and load the sarcoplasmic reticulum and likely contributes to the altered electromechanical properties of the hypertrophied heart.Rationale: Pathological increases in cardiac afterload result in myocyte hypertrophy with changes in myocyte electrical and mechanical phenotype. Remodeling of contractile and signaling Ca 2+ occurs in pathological hypertrophy and is central to myocyte remodeling. Stromal Interaction Molecule 1 (STIM1) regulates Ca 2+ signaling in many cell types by sensing low endoplasmic reticular Ca 2+ levels and then coupling to plasma membrane Orai channels to induce a Ca 2+ influx pathway. Previous reports suggest that STIM1 may play a role in cardiac hypertrophy but its role in electrical and mechanical phenotypic alterations are not well understood. Objective: To define the contributions of STIM1-mediated Ca 2+ influx on electrical and mechanical properties of normal and diseased myocytes, and to determine if Orai channels are obligatory partners for STIM1 in these processes using a clinically relevant large animal model of hypertrophy. Methods and Results: Cardiac hypertrophy was induced by slow progressive pressure overload in adult cats. Hypertrophied myocytes had increased STIM1 expression and activity, which correlated with altered Ca 2+ handling and action potential (AP) prolongation. Exposure of hypertrophied myocytes to the Orai channel blocker BTP2 caused in a reduction of AP duration and reduced diastolic Ca 2+ spark rate. BTP2 had no effect on normal myocytes. Forced expression of STIM1 in cultured adult feline ventricular myocytes increased diastolic spark rate and prolonged AP duration. STIM1 expression produced an increase in the amount of Ca 2+ stored within the sarcoplasmic reticulum and activated Ca 2+ /calmodulin-dependent protein kinase II. STIM1 expression also increased spark rates and induced spontaneous APs. STIM1 effects were eliminated by either BTP2 or by co-expression of a dominant negative Orai construct. Conclusions: STIM1 can associate with Orai in cardiac myocytes to produce a Ca 2+ influx pathway that can prolong the AP duration and load the SR and likely contributes to the altered electromechanical properties of the hypertrophied heart.

Cortical Bone Stem Cell Therapy Preserves Cardiac Structure and Function After Myocardial InfarctionNovelty and Significance

Rationale: Cortical bone stem cells (CBSCs) have been shown to reduce ventricular remodeling and improve cardiac function in a murine myocardial infarction (MI) model. These effects were superior to other stem cell types that have been used in recent early-stage clinical trials. However, CBSC efficacy has not been tested in a preclinical large animal model using approaches that could be applied to patients. Objective: To determine whether post-MI transendocardial injection of allogeneic CBSCs reduces pathological structural and functional remodeling and prevents the development of heart failure in a swine MI model. Methods and Results: Female Göttingen swine underwent left anterior descending coronary artery occlusion, followed by reperfusion (ischemia–reperfusion MI). Animals received, in a randomized, blinded manner, 1:1 ratio, CBSCs (n=9; 2×107 cells total) or placebo (vehicle; n=9) through NOGA-guided transendocardial injections. 5–ethynyl–2′deoxyuridine (EdU)—a thymidine analog—containing minipumps were inserted at the time of MI induction. At 72 hours (n=8), initial injury and cell retention were assessed. At 3 months post-MI, cardiac structure and function were evaluated by serial echocardiography and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72 hours post-MI but had no effect on initial cardiac injury or structure. At 3 months, CBSC-treated hearts had significantly reduced scar size, smaller myocytes, and increased myocyte nuclear density. Noninvasive echocardiographic measurements showed that left ventricular volumes and ejection fraction were significantly more preserved in CBSC-treated hearts, and invasive hemodynamic measurements documented improved cardiac structure and functional reserve. The number of EdU+ cardiac myocytes was increased in CBSC- versus vehicle- treated animals. Conclusions: CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves left ventricular functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction.

The Anti-Cancer Multikinase Inhibitor Sorafenib Impairs Cardiac Contractility by Reducing Phospholamban Phosphorylation and Sarcoplasmic Calcium Transients

Tyrosine-kinase inhibitors (TKIs) have revolutionized cancer therapy in recent years. Although more targeted than conventional chemotherapy, TKIs exhibit substantial cardiotoxicity, often manifesting as hypertension or heart failure. Here, we assessed myocyte intrinsic cardiotoxic effects of the TKI sorafenib and investigated underlying alterations of myocyte calcium homeostasis. We found that sorafenib reversibly decreased developed force in auxotonically contracting human myocardia (3 µM: −25 ± 4%, 10 µM: −29 ± 7%, 30 µM: −43 ± 12%, p < 0.01), reduced peak cytosolic calcium concentrations in isolated cardiomyocytes (10 µM: 52 ± 8.1% of baseline, p < 0.001), and slowed cytosolic calcium removal kinetics (RT50, RT10, Tau, p < 0.05). Beta-adrenergic stimulation induced augmentation of calcium transient (CaT) amplitude was attenuated in sorafenib-treated cells (2.7 ± 0.3-fold vs. 3.6 ± 0.2-fold in controls, p < 0.001). Sarcoplasmic reticulum (SR) calcium content was reduced to 67 ± 4% (p < 0.01), and SR calcium re-uptake slowed (p < 0.05). Sorafenib significantly reduced serine 16 phosphorylation of phospholamban (PLN, p < 0.05), while PLN threonine 17 and CaMKII (T286) phosphorylation were not altered. Our data demonstrate that sorafenib acutely impairs cardiac contractility by reducing S16 PLN phosphorylation, leading to reduced SR calcium content, CaT amplitude, and slowed cytosolic calcium removal. These results indicate myocyte intrinsic cardiotoxicity irrespective of effects on the vasculature and chronic cardiac remodeling.

Echocardiographic Strain Analysis for the Early Detection of Left Ventricular Systolic/Diastolic Dysfunction and Dyssynchrony in a Mouse Model of Physiological Aging

Abstract Heart disease is the leading cause of hospitalization and death worldwide, severely affecting health care costs. Aging is a significant risk factor for heart disease, and the senescent heart is characterized by structural and functional changes including diastolic and systolic dysfunction as well as left ventricular (LV) dyssynchrony. Speckle tracking–based strain echocardiography (STE) has been shown as a noninvasive, reproducible, and highly sensitive methodology to evaluate LV function in both animal models and humans. Herein, we describe the efficiency of this technique as a comprehensive and sensitive method for the detection of age-related cardiac dysfunction in mice. Compared with conventional echocardiographic measurements, radial and longitudinal strain, and reverse longitudinal strain were able to detect subtle changes in systolic and diastolic cardiac function in mice at an earlier time point during aging. Additionally, the data show a gradual and consistent decrease with age in regional contractility throughout the entire LV, in both radial and longitudinal axes. Furthermore, we observed that LV segmental dyssynchrony in longitudinal axis reliably differentiated between aged and young mice. Therefore, we propose the use of echocardiographic strain as a highly sensitive and accurate technology enabling and evaluating the effect of new treatments to fight age-induced cardiac disease.

A Feline HFpEF Model with Pulmonary Hypertension and Compromised Pulmonary Function

Heart Failure with preserved Ejection Fraction (HFpEF) represents a major public health problem. The causative mechanisms are multifactorial and there are no effective treatments for HFpEF, partially attributable to the lack of well-established HFpEF animal models. We established a feline HFpEF model induced by slow-progressive pressure overload. Male domestic short hair cats (n = 20), underwent either sham procedures (n = 8) or aortic constriction (n = 12) with a customized pre-shaped band. Pulmonary function, gas exchange, and invasive hemodynamics were measured at 4-months post-banding. In banded cats, echocardiography at 4-months revealed concentric left ventricular (LV) hypertrophy, left atrial (LA) enlargement and dysfunction, and LV diastolic dysfunction with preserved systolic function, which subsequently led to elevated LV end-diastolic pressures and pulmonary hypertension. Furthermore, LV diastolic dysfunction was associated with increased LV fibrosis, cardiomyocyte hypertrophy, elevated NT-proBNP plasma levels, fluid and protein loss in pulmonary interstitium, impaired lung expansion, and alveolar-capillary membrane thickening. We report for the first time in HFpEF perivascular fluid cuff formation around extra-alveolar vessels with decreased respiratory compliance. Ultimately, these cardiopulmonary abnormalities resulted in impaired oxygenation. Our findings support the idea that this model can be used for testing novel therapeutic strategies to treat the ever growing HFpEF population.

Protein Kinase C Inhibition With Ruboxistaurin Increases Contractility and Reduces Heart Size in a Swine Model of Heart Failure With Reduced Ejection Fraction

Visual Abstract

Remodeling of repolarization and arrhythmia susceptibility in a myosin-binding protein C knockout mouse model

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases and among the leading causes of sudden cardiac death (SCD) in the young. The cellular mechanisms leading to SCD in HCM are not well known. Prolongation of the action potential (AP) duration (APD) is a common feature predisposing hypertrophied hearts to SCD. Previous studies have explored the roles of inward Na+ and Ca2+ in the development of HCM, but the role of repolarizing K+ currents has not been defined. The objective of this study was to characterize the arrhythmogenic phenotype and cellular electrophysiological properties of mice with HCM, induced by myosin-binding protein C (MyBPC) knockout (KO), and to test the hypothesis that remodeling of repolarizing K+ currents causes APD prolongation in MyBPC KO myocytes. We demonstrated that MyBPC KO mice developed severe hypertrophy and cardiac dysfunction compared with wild-type (WT) control mice. Telemetric electrocardiographic recordings of awake mice revealed prolongation of the corrected QT interval in the KO compared with WT control mice, with overt ventricular arrhythmias. Whole cell current- and voltage-clamp experiments comparing KO with WT mice demonstrated ventricular myocyte hypertrophy, AP prolongation, and decreased repolarizing K+ currents. Quantitative RT-PCR analysis revealed decreased mRNA levels of several key K+ channel subunits. In conclusion, decrease in repolarizing K+ currents in MyBPC KO ventricular myocytes contributes to AP and corrected QT interval prolongation and could account for the arrhythmia susceptibility.NEW & NOTEWORTHY Ventricular myocytes isolated from the myosin-binding protein C knockout hypertrophic cardiomyopathy mouse model demonstrate decreased repolarizing K+ currents and action potential and QT interval prolongation, linking cellular repolarization abnormalities with arrhythmia susceptibility and the risk for sudden cardiac death in hypertrophic cardiomyopathy.

Nuquantus: Machine learning software for the characterization and quantification of cell nuclei in complex immunofluorescent tissue images

Determination of fundamental mechanisms of disease often hinges on histopathology visualization and quantitative image analysis. Currently, the analysis of multi-channel fluorescence tissue images is primarily achieved by manual measurements of tissue cellular content and sub-cellular compartments. Since the current manual methodology for image analysis is a tedious and subjective approach, there is clearly a need for an automated analytical technique to process large-scale image datasets. Here, we introduce Nuquantus (Nuclei quantification utility software) - a novel machine learning-based analytical method, which identifies, quantifies and classifies nuclei based on cells of interest in composite fluorescent tissue images, in which cell borders are not visible. Nuquantus is an adaptive framework that learns the morphological attributes of intact tissue in the presence of anatomical variability and pathological processes. Nuquantus allowed us to robustly perform quantitative image analysis on remodeling cardiac tissue after myocardial infarction. Nuquantus reliably classifies cardiomyocyte versus non-cardiomyocyte nuclei and detects cell proliferation, as well as cell death in different cell classes. Broadly, Nuquantus provides innovative computerized methodology to analyze complex tissue images that significantly facilitates image analysis and minimizes human bias.