Gunnar Klein

A Cathepsin D-Cleaved 16 kDa Form of Prolactin Mediates Postpartum Cardiomyopathy

Postpartum cardiomyopathy (PPCM) is a disease of unknown etiology and exposes women to high risk of mortality after delivery. Here, we show that female mice with a cardiomyocyte-specific deletion of stat3 develop PPCM. In these mice, cardiac cathepsin D (CD) expression and activity is enhanced and associated with the generation of a cleaved antiangiogenic and proapoptotic 16 kDa form of the nursing hormone prolactin. Treatment with bromocriptine, an inhibitor of prolactin secretion, prevents the development of PPCM, whereas forced myocardial generation of 16 kDa prolactin impairs the cardiac capillary network and function, thereby recapitulating the cardiac phenotype of PPCM. Myocardial STAT3 protein levels are reduced and serum levels of activated CD and 16 kDa prolactin are elevated in PPCM patients. Thus, a biologically active derivative of the pregnancy hormone prolactin mediates PPCM, implying that inhibition of prolactin release may represent a novel therapeutic strategy for PPCM.

Catheter-Based Renal Nerve Ablation and Centrally Generated Sympathetic Activity in Difficult-to-Control Hypertensive Patients Prospective Case Series

Endovascular renal nerve ablation has been developed to treat resistant hypertension. In addition to lowering efferent renal sympathetic activation, the intervention may attenuate central sympathetic outflow through decreased renal afferent nerve traffic, as evidenced by a recent case report. We tested the hypothesis in 12 nonpreselected patients with difficult-to-control hypertension (aged 45–74 years) admitted for renal nerve ablation. All patients received ≥3 antihypertensive medications at full doses, including a diuretic. Electrocardiogram, respiration, brachial and finger arterial blood pressure, and muscle sympathetic nerve activity were recorded before and 3 to 6 months after renal nerve ablation. Heart rate and blood pressure variability were analyzed in the time and frequency domain. Pharmacological baroreflex slopes were determined using the modified Oxford bolus technique. Resting heart rate was 61±3 bpm before and 58±2 bpm after ablation (P=0.4). Supine blood pressure was 157±7/85±4 mm Hg before and 157±6/85±4 mm Hg after ablation (P=1.0). Renal nerve ablation did not change resting muscle sympathetic nerve activity (before, 34±2 bursts per minute; after, 32±3 bursts per minute P=0.6), heart rate variability, or blood pressure variability. Pharmacological baroreflex control of heart rate and muscle sympathetic nerve activity did not change. We conclude that reduced central sympathetic inhibition may be the exception rather than the rule after renal nerve ablation in unselected patients with difficult-to-control arterial hypertension.

Efficacy of pulmonary vein isolation by cryoballoon ablation in patients with paroxysmal atrial fibrillation.

BACKGROUND Radiofrequency ablation of pulmonary veins (PVs) has emerged as an effective treatment for patients with paroxysmal atrial fibrillation (AF). However, serious complications raise concern about an even wider application. In terms of safety, cryoenergy has advantages compared with radiofrequency. A new cryoenergy balloon catheter has been recently developed to make AF ablation shorter and safer. OBJECTIVE The purpose of this study was to test the 6-month efficacy of this new device for ablation of paroxysmal AF. METHODS Twenty-one patients with highly symptomatic paroxysmal AF, normal left atrial size, and frequent episodes of AF were included. All PVs were targeted during cryoballoon ablation. Patients received 24-hour Holter electrocardiograms (ECGs) and event recorder during follow-up after 1, 3, and 6 months. RESULTS A total of 81 (95%) of 85 PVs could be completely isolated with a single-balloon technique. Procedure time was 165 +/- 35 minutes, and fluoroscopy time was 39 +/- 9 minutes. After 6 months, 86% of the patients were free of symptomatic AF. In two of three patients with recurrence of AF, complete PV isolation has not been achieved initially. After a second procedure (1.04 procedures per patient), 90% of the patients were free of symptomatic AF. Three phrenic nerve palsies occurred during ablation of the right superior PV; two completely resolved after 6 and 9 months, and one is still persisting after 2 months. CONCLUSION This is the first study that reports the results of the new cryoballoon AF ablation approach showing 86% freedom from AF recurrence after 6 months. Cryoballoon PV ablation promises to be effective for patients with paroxysmal AF and normally sized left atria.

Reversal of IFN-γ, oxLDL and prolactin serum levels correlate with clinical improvement in patients with peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is characterized by acute onset of heart failure of unknown aetiology. We aimed to identify mechanisms involved in initiation and progression of the disease.

Role of interleukin-6 for LV remodeling and survival after experimental myocardial infarction

Circulating levels of interleukin (IL)‐6 are elevated after myocardial infarction (MI) and associated with increased morbidity and mortality. Its myocardial expression post‐MI suggests a pathophysiological role in this condition. To explore the role of endogenous IL‐6, we analyzed MI size, left ventricular (LV) remodeling, and mortality after permanent coronary ligation in IL‐6 knockout mice (IL‐6−/−) and wild‐type controls (WT). Six weeks after MI, IL‐6−/− and WT had similar mortality rates, MI sizes, LV remodeling, and LV dysfunction in vivo, determined by catheterization. Infarct size 24 h post‐MI, shown by 2,3,5‐triphenyltetrazolium chloride (TTC) staining, was similar at 24 h. Treatment with exogenous IL‐6 did not alter MI size in WT. Infarction resulted in marked phosphorylation of STAT3, without differences between genotypes. Leukemia inhibitory factor (LIF) protein was increased 48 h post‐MI in IL‐6−/−, and angiotensin II and AT1 receptor (AT1R) protein were strongly increased in IL‐6−/− baseline and post‐MI, suggesting compensatory up‐regulation. Lack of IL‐6 does not affect long‐term MI size or LV function, remodeling, and survival. In mice lacking IL‐6, other members of the IL‐6 family such as LIF and other factors signaling via JAK/STAT such as angiotensin may act in a compensatory manner to activate the JAK/STAT pathway, thereby maintaining STAT3 phosphorylation, which is crucial for the cellular effects of IL‐6 cytokines.

Regulation of Proangiogenic Factor CCN1 in Cardiac Muscle Impact of Ischemia, Pressure Overload, and Neurohumoral Activation

Background—CCN1, a potent proangiogenic factor, is induced in the vasculature by tissue injury, angiotensin II (Ang II), and growth factor stimulation. Because these conditions occur in myocardial ischemia and pressure overload, we investigated the regulation of CCN1 in cardiomyocytes in vitro and in the heart in vivo. Methods and Results—Ang II, signaling via the angiotensin type 1 (AT1) receptor, and &agr;1-adrenergic stimulation with phenylephrine induced CCN1 expression in ventricular cardiomyocytes isolated from 1- to 3-day-old rats. Cell culture supernatant of Ang II–treated cardiomyocytes induced migration of smooth muscle cells, which was abolished by neutralizing antibody to CCN1. Ang II– and phenylephrine-mediated induction of CCN1 expression in cardiomyocytes was completely abolished by inhibition of MEK/extracellular signal–regulated kinases (ERK) or protein kinase C (PKC). Likewise, mechanical stretch induced CCN1 expression in cardiomyocytes, an effect that was prevented by AT1 receptor blockade or PKC inhibition. Similarly, pressure overload in vivo upregulated myocardial CCN1 expression levels via AT1 receptor– and PKC-dependent mechanisms. After myocardial infarction in mice, CCN1 expression was strongly induced in both ischemic and remote left ventricular myocardium. Marked CCN1 protein expression was noted in cardiomyocytes of patients with end-stage ischemic cardiomyopathy but was almost absent in nonfailing human myocardium. Conclusions—Pressure overload, ischemia, and neurohormonal factors, such as Ang II or &agr;1-adrenergic stimuli, induce myocardial expression of CCN1, a potent proangiogenic factor, supporting the notion that CCN1 may play an important role in the adaptation of the heart to cardiovascular stress.

Increased Collagen Deposition and Diastolic Dysfunction but Preserved Myocardial Hypertrophy After Pressure Overload in Mice Lacking PKCε

Overexpression and activation of protein kinase C-&egr; (PKC&egr;) results in myocardial hypertrophy. However, these observations do not establish that PKC&egr; is required for the development of myocardial hypertrophy. Thus, we subjected PKC&egr;-knockout (KO) mice to a hypertrophic stimulus by transverse aortic constriction (TAC). KO mice show normal cardiac morphology and function. TAC caused similar cardiac hypertrophy in KO and wild-type (WT) mice. However, KO mice developed more interstitial fibrosis and showed enhanced expression of collagen I&agr;1 and collagen III after TAC associated with diastolic dysfunction, as assessed by tissue Doppler echocardiography (Ea/Aa after TAC: WT 2.1±0.3 versus KO 1.0±0.2; P<0.05). To explore underlying mechanisms, we analyzed the left ventricular (LV) expression pattern of additional PKC isoforms (ie, PKC&agr;, PKC&bgr;, and PKC&dgr;). After TAC, expression and activation of PKC&dgr; protein was increased in KO LVs. Moreover, KO LVs displayed enhanced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), whereas p42/p44–MAPK activation was attenuated. Under stretch, cultured KO fibroblasts showed a 2-fold increased collagen I&agr;1 (col I&agr;1) expression, which was prevented by PKC&dgr; inhibitor rottlerin or by p38 MAPK inhibitor SB 203580. In conclusion, PKC&egr; is not required for the development of a pressure overload–induced myocardial hypertrophy. Lack of PKC&egr; results in upregulation of PKC&dgr; and promotes activation of p38 MAPK and JNK, which appears to compensate for cardiac hypertrophy, but in turn, is associated with increased collagen deposition and impaired diastolic function.

Continuous Glycoprotein-130–Mediated Signal Transducer and Activator of Transcription-3 Activation Promotes Inflammation, Left Ventricular Rupture, and Adverse Outcome in Subacute Myocardial Infarction

Background— In patients with myocardial infarction, high serum levels of interleukin-6 cytokines predict a poor outcome. The common receptor of interleukin-6 cytokines, glycoprotein-130 (gp130), signals via janus kinase/signal transducer and activator of transcription (STAT), cytoplasmic protein tyrosine phosphatase/extracellular signal-regulated kinase, and phosphoinositide-3-kinase/Akt pathways, and the regulation of these pathways depends at least in part on the gp130 tyrosine-757 residue. By analyzing cardiomyocyte-specific gp130Y757F mutant mice, we investigated the effect of disturbed gp130 signaling after myocardial infarction. Methods and Results— The cardiomyocyte-restricted &agr;-myosin heavy chain-Cre-recombinase-loxP system was used to generate mice with gp130Y757F mutant cardiomyocytes (&agr;MHC-Cretg/−;gp130fl/Y757F [Y757F]); all other cells carried at least 1 functional gp130 gene, ensuring normal gp130 signaling. Y757F mice displayed normal cardiac function and morphology at 3 months of age comparable to their nonmutant littermates. In response to myocardial infarction, Y757F mice displayed higher mortality associated with increased left ventricular rupture rate, sustained cardiac inflammation, and heart failure. These adverse effects were associated with prolonged and enhanced STAT3 activation and increased expression of interleukin-6 and of the complement-activating mannose-binding lectin C. Pharmacological inhibition of the complement system by cobra venom factor attenuated inflammation, prevented left ventricular rupture, and improved cardiac function in Y757F mice. Stronger effects were observed with a genetic reduction of STAT3 (STAT3flox/+) restricted to cardiomyocytes in Y757F mice, which prevented extensive upregulation of interleukin-6, complement activation, and sustained inflammation and lowered left ventricular rupture rate, heart failure, and mortality in subacute myocardial infarction. Conclusion— Impaired downregulation of gp130-mediated STAT3 activation in subacute infarction promotes cardiac inflammation, adverse remodeling, and heart failure, suggesting a potential causative role of high interleukin-6 serum levels after myocardial infarction.

Single L-type Ca2+ channel regulation by cGMP-dependent protein kinase type I in adult cardiomyocytes from PKG I transgenic mice

OBJECTIVE Calcium entry via the L-type Ca(2+) channel (LTCC) is crucial for excitation-contraction (EC) coupling and activation of Ca(2+)-dependent signal transduction pathways in cardiac myocytes. Both nitric oxide (NO), signaling via cGMP, and acetylcholine, signaling via the muscarinic receptor, have been identified as negative regulators of beta-adrenoreceptor-stimulated LTCC activity in cardiac myocytes. METHODS To examine the potential role of cGMP-dependent protein kinase type I (PKG I) in the inhibitory effects of NO/cGMP and the muscarinic receptor on LTCC activity, we generated transgenic (TG) mice overexpressing PKG I selectively in cardiac myocytes under the control of the alpha-myocin heavy chain promoter. Single LTCC-gating properties were assessed in isolated ventricular myocytes from adult wild-type (WT) and PKG I transgenic (TG) mice. RESULTS Basal LTCC activity (peak average current, mean open probability, mean availability) was significantly decreased by the nitric oxide donor DEA-NO (0.1 micromol/l) and the cGMP-analog 8-Br-cGMP (1 mmol/l) in TG but not in WT cardiac myocytes. Conversely, muscarinic (carbachol, 1 micromol/l) stimulation had no significant effect on basal LTCC activity in either WT or TG cardiac myocytes. beta-Adrenergic stimulation with isoproterenol (1 micromol/l) increases single LTCC activity in WT and TG cardiac myocytes to the same extent. The inhibitory effects of DEA-NO and 8-Br-cGMP on isoproterenol activation of the LTCC current were significantly enhanced in TG as compared to WT cardiac myocytes. By contrast, carbachol inhibition of isoproterenol-stimulated single LTCC activity was not enhanced in TG cardiac myocytes. CONCLUSION Transgenic overexpression of PKG I augments NO/cGMP inhibition but not muscarinic inhibition of single LTCC activity, indicating that PKG I is a downstream target for NO/cGMP, but not the muscarinic receptor in adult cardiac myocytes.

Evaluation of left ventricular diastolic function by pulsed Doppler tissue imaging in mice.

BACKGROUND Diastolic left ventricular (LV) function is commonly characterized by transmitral flow pattern in human beings. Recently, Doppler tissue imaging (DTI) was introduced to evaluate diastolic function. The aim of our study was to validate DTI in the evaluation of diastolic function in mice. METHODS We measured indices of diastolic function using pulsed DTI, and transmitral Doppler and LV pressure and its maximal rate of decrease (LVdP/dt(min)), before and 4 weeks after aortic banding in C57BL/6 mice. RESULTS Peak early diastolic velocity and ratio of peak early-to-late filling velocities, both measured by DTI, were significantly reduced after banding, thereby indicating diastolic dysfunction. Diastolic dysfunction was confirmed by impaired LV dP/dt(min), decreased transmitral early filling velocity, and transmitral early-to-late filling velocity ratio using transmitral Doppler. CONCLUSION DTI detects diastolic dysfunction caused by chronic pressure overload in mice after aortic banding. DTI is suggested to be implemented as part of routine mouse echocardiography for evaluation of LV diastolic function.