Bernhard Unsöld

Differential Cardiac Remodeling in Preload Versus Afterload

Background— Hemodynamic load regulates myocardial function and gene expression. We tested the hypothesis that afterload and preload, despite similar average load, result in different phenotypes. Methods and Results— Afterload and preload were compared in mice with transverse aortic constriction (TAC) and aortocaval shunt (shunt). Compared with sham mice, 6 hours after surgery, systolic wall stress (afterload) was increased in TAC mice (+40%; P<0.05), diastolic wall stress (preload) was increased in shunt (+277%; P<0.05) and TAC mice (+74%; P<0.05), and mean total wall stress was similarly increased in TAC (69%) and shunt mice (67%) (P=NS, TAC versus shunt; each P<0.05 versus sham). At 1 week, left ventricular weight/tibia length was significantly increased by 22% in TAC and 29% in shunt mice (P=NS, TAC versus shunt). After 24 hours and 1 week, calcium/calmodulin-dependent protein kinase II signaling was increased in TAC. This resulted in altered calcium cycling, including increased L-type calcium current, calcium transients, fractional sarcoplasmic reticulum calcium release, and calcium spark frequency. In shunt mice, Akt phosphorylation was increased. TAC was associated with inflammation, fibrosis, and cardiomyocyte apoptosis. The latter was significantly reduced in calcium/calmodulin-dependent protein kinase IIΔ-knockout TAC mice. A total of 157 mRNAs and 13 microRNAs were differentially regulated in TAC versus shunt mice. After 8 weeks, fractional shortening was lower and mortality was higher in TAC versus shunt mice. Conclusions— Afterload results in maladaptive fibrotic hypertrophy with calcium/calmodulin-dependent protein kinase II–dependent altered calcium cycling and apoptosis. Preload is associated with Akt activation without fibrosis, little apoptosis, better function, and lower mortality. This indicates that different loads result in distinct phenotype differences that may require specific pharmacological interventions.

Generation of Functional Cardiomyocytes From Adult Mouse Spermatogonial Stem Cells

Stem cell–based therapy is a promising approach for the treatment of heart failure. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Recently, we reported the successful establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. These cultured maGSCs show phenotypic characteristics similar to ESCs and can spontaneously differentiate into cells from all 3 germ layers. In the present study, we used the hanging drop method to differentiate maGSCs into cardiomyocytes and analyzed their functional properties. Differentiation efficiency of beating cardiomyocytes from maGSCs was similar to that from ESCs. The maGSC-derived cardiomyocytes expressed cardiac-specific L-type Ca2+ channels and responded to Ca2+ channel–modulating drugs. Cx43 was expressed at cell-to-cell contacts in cardiac clusters, and fluorescence recovery after photobleaching assay showed the presence of functional gap junctions among cardiomyocytes. Action potential analyses demonstrated the presence of pacemaker-, ventricle-, atrial-, and Purkinje-like cardiomyocytes. Stimulation with isoproterenol resulted in a significant increase in beating frequency, whereas the addition of cadmium chloride abolished spontaneous electrical activity. Confocal microscopy analysis of intracellular Ca2+ in maGSC-derived cardiomyocytes showed that calcium increased periodically throughout the cell in a homogenous fashion, pointing to a fine regulated Ca2+ release from intracellular Ca2+ stores. By using line-scan mode, we found rhythmic Ca2+ transients. Furthermore, we transplanted maGSCs into normal hearts of mice and found that maGSCs were able to proliferate and differentiate. No tumor formation was found up to 1 month after cell transplantation. Taken together, we believe that maGSCs provide a new source of distinct types of cardiomyocytes for basic research and potential therapeutic application.

Constitutively active phosphatase inhibitor-1 improves cardiac contractility in young mice but is deleterious after catecholaminergic stress and with aging

Phosphatase inhibitor-1 (I-1) is a distal amplifier element of beta-adrenergic signaling that functions by preventing dephosphorylation of downstream targets. I-1 is downregulated in human failing hearts, while overexpression of a constitutively active mutant form (I-1c) reverses contractile dysfunction in mouse failing hearts, suggesting that I-1c may be a candidate for gene therapy. We generated mice with conditional cardiomyocyte-restricted expression of I-1c (referred to herein as dTGI-1c mice) on an I-1-deficient background. Young adult dTGI-1c mice exhibited enhanced cardiac contractility but exaggerated contractile dysfunction and ventricular dilation upon catecholamine infusion. Telemetric ECG recordings revealed typical catecholamine-induced ventricular tachycardia and sudden death. Doxycycline feeding switched off expression of cardiomyocyte-restricted I-1c and reversed all abnormalities. Hearts from dTGI-1c mice showed hyperphosphorylation of phospholamban and the ryanodine receptor, and this was associated with an increased number of catecholamine-induced Ca2+ sparks in isolated myocytes. Aged dTGI-1c mice spontaneously developed a cardiomyopathic phenotype. These data were confirmed in a second independent transgenic mouse line, expressing a full-length I-1 mutant that could not be phosphorylated and thereby inactivated by PKC-alpha (I-1S67A). In conclusion, conditional expression of I-1c or I-1S67A enhanced steady-state phosphorylation of 2 key Ca2+-regulating sarcoplasmic reticulum enzymes. This was associated with increased contractile function in young animals but also with arrhythmias and cardiomyopathy after adrenergic stress and with aging. These data should be considered in the development of novel therapies for heart failure.

Phosphatase inhibitor-1-deficient mice are protected from catecholamine-induced arrhythmias and myocardial hypertrophy

AIMS Phosphatase inhibitor-1 (I-1) is a conditional amplifier of beta-adrenergic signalling downstream of protein kinase A by inhibiting type-1 phosphatases only in its PKA-phosphorylated form. I-1 is downregulated in failing hearts and thus contributes to beta-adrenergic desensitization. It is unclear whether this should be viewed as a predominantly adverse or protective response. METHODS AND RESULTS We generated transgenic mice with cardiac-specific I-1 overexpression (I-1-TG) and evaluated cardiac function and responses to catecholamines in mice with targeted disruption of the I-1 gene (I-1-KO). Both groups were compared with their wild-type (WT) littermates. I-1-TG developed cardiac hypertrophy and mild dysfunction which was accompanied by a substantial compensatory increase in PP1 abundance and activity, confounding cause-effect relationships. I-1-KO had normal heart structure with mildly reduced sensitivity, but unchanged maximal contractile responses to beta-adrenergic stimulation, both in vitro and in vivo. Notably, I-1-KO were partially protected from lethal catecholamine-induced arrhythmias and from hypertrophy and dilation induced by a 7 day infusion with the beta-adrenergic agonist isoprenaline. Moreover, I-1-KO exhibited a partially preserved acute beta-adrenergic response after chronic isoprenaline, which was completely absent in similarly treated WT. At the molecular level, I-1-KO showed lower steady-state phosphorylation of the cardiac ryanodine receptor/Ca(2+) release channel and the sarcoplasmic reticulum (SR) Ca(2+)-ATPase-regulating protein phospholamban. These alterations may lower the propensity for diastolic Ca(2+) release and Ca(2+) uptake and thus stabilize the SR and account for the protection. CONCLUSION Taken together, loss of I-1 attenuates detrimental effects of catecholamines on the heart, suggesting I-1 downregulation in heart failure as a beneficial desensitization mechanism and I-1 inhibition as a potential novel strategy for heart failure treatment.

Parthenogenetic stem cells for tissue-engineered heart repair

Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.

Telethonin Deficiency Is Associated With Maladaptation to Biomechanical Stress in the Mammalian Heart

Rationale: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique &bgr;-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonins in vivo function. Objective: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation. Methods and Results: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin–titin cross-links via &agr;-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis (“mechanoptosis”). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts. Conclusions: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.

Effects of large volume, ice-cold intravenous fluid infusion on respiratory function in cardiac arrest survivors

International guidelines for cardiopulmonary resuscitation recommend mild hypothermia (32-34 degrees C) for 12-24h in comatose survivors of cardiac arrest. To induce therapeutic hypothermia a variety of external and intravascular cooling devices are available. A cheap and effective method for inducing hypothermia is the infusion of large volume, ice-cold intravenous fluid. There are concerns regarding the effects of rapid infusion of large volumes of fluid on respiratory function in cardiac arrest survivors. We have retrospectively studied the effects of high volume cold fluid infusion on respiratory function in 52 resuscitated cardiac arrest patients. The target temperature of 32-34 degrees C was achieved after 4.1+/-0.5h (cooling rate 0.48 degrees C/h). During this period 3427+/-210 mL ice-cold fluid was infused. Despite significantly reduced LV-function (EF 35.8+/-2.2%) the respiratory status of these patients did not deteriorate significantly. On intensive care unit admission the mean PaO(2) was 231.4+/-20.6 mmHg at a F(i)O(2) of 0.82+/-0.03 (PaO(2)/F(i)O(2)=290.0+/-24.1) and a PEEP level of 7.14+/-0.31 mbar. Until reaching the target temperature of <or=34 degrees C the F(i)O(2) could be significantly reduced to 0.63+/-0.03 with unchanged PEEP level (7.23+/-0.36 mbar). Under these conditions the PaO(2)/F(i)O(2) ratio slightly decreased to 247.5+/-18.5 (P=0.0893). Continuing the saline infusion to achieve a body temperature of 33 degrees C, the F(i)O(2) could be further reduced with unchanged PEEP. The infusion of large volume, ice-cold fluid is an effective and inexpensive method for inducing therapeutic hypothermia. Resuscitation from cardiac arrest is associated with a deterioration in respiratory function. The infusion of large volumes of cold fluid does not cause a statistically significant further deterioration in respiratory function. A larger, randomized and prospective study is required to assess the efficacy and safety of ice-cold fluid infusion for the induction of therapeutic hypothermia.

Exercise intolerance and systemic manifestations of pulmonary emphysema in a mouse model

BackgroundSystemic effects of chronic obstructive pulmonary disease (COPD) significantly contribute to severity and mortality of the disease. We aimed to develop a COPD/emphysema model exhibiting systemic manifestations of the disease.MethodsFemale NMRI mice were treated 5 times intratracheally with porcine pancreatic elastase (emphysema) or phosphate-buffered saline (control). Emphysema severity was quantified histologically by mean linear intercept, exercise tolerance by treadmill running distance, diaphragm dysfunction using isolated muscle strips, pulmonary hypertension by measuring right ventricular pressure, and neurohumoral activation by determining urinary norepinephrine concentration.ResultsMean linear intercept was higher in emphysema (260.7 ± 26.8 μm) than in control lungs (24.7 ± 1.7 μm). Emphysema mice lost body weight, controls gained weight. Running distance was shorter in emphysema than in controls. Diaphragm muscle length was shorter in controls compared to emphysema. Fatigue tests of muscle strips revealed impaired relaxation in emphysema diaphragms. Maximum right ventricular pressure and norepinephrine were elevated in emphysema compared to controls. Linear correlations were observed between running distance changes and intercept, right ventricular weight, norepinephrine, and diaphragm length.ConclusionThe elastase mouse model exhibited severe emphysema with consecutive exercise limitation, and neurohumoral activation. The model may deepen our understanding of systemic aspects of COPD.

Sensing Cardiac Electrical Activity With a Cardiac Myocyte Targeted Optogenetic Voltage Indicator

RATIONALE Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. OBJECTIVE To test the hypothesis that cardiac myocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electric activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell-derived cardiac myocytes. METHODS AND RESULTS We first generated mice with cardiac myocyte-restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch clamping. The use of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in induced pluripotent stem cell-derived cardiac myocytes. CONCLUSIONS We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof of concept for its use in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.

Melusin protects from cardiac rupture and improves functional remodelling after myocardial infarction

AIMS Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicentre approach. METHODS AND RESULTS Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusins overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P = 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein expression in the infarcted area. At 2 weeks after MI, melusin overexpression conferred a favourable adaptive remodelling characterized by reduced left ventricle dilatation and better preserved contractility in the presence of a comparable degree of hypertrophy. Adaptive remodelling in melusin TG mice was characterized by reduced apoptosis and fibrosis as well as increased cardiomyocyte contractility. CONCLUSIONS Consistent with its function as a chaperone protein, melusin overexpression exerts a dual protective action following MI reducing an array of maladaptive processes. In the early phase after MI, reduced inflammation and myocyte remodelling protect against cardiac rupture. Chronically, reduced myocyte loss and matrix remodelling, with preserved myocyte contractility, confer adaptive LV remodelling.