Tim Seidler

Increased Sarcoplasmic Reticulum Calcium Leak but Unaltered Contractility by Acute CaMKII Overexpression in Isolated Rabbit Cardiac Myocytes

The predominant cardiac Ca2+/calmodulin-dependent protein kinase (CaMK) is CaMKII&dgr;. Here we acutely overexpress CaMKII&dgr;C using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKII&dgr;C transgenic mice. CaMKII&dgr;C protein expression and activation state (autophosphorylation) were increased 5- to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKII&dgr;C versus LacZ expressing myocytes. However, the contraction–frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced (&tgr;0.5Hz/&tgr;3Hz=2.14±0.10 versus 1.87±0.10), and peak Ca2+ current (ICa) was increased by 31% (−7.1±0.5 versus −5.4±0.5 pA/pF, P<0.05). Ca2+ transient amplitude was not significantly reduced (−27%, P=0.22), despite dramatically reduced sarcoplasmic reticulum (SR) Ca2+ content (41%; P<0.05). Thus fractional SR Ca2+ release was increased by 60% (P<0.05). Diastolic SR Ca2+ leak assessed by Ca2+ spark frequency (normalized to SR Ca2+ load) was increased by 88% in CaMKII&dgr;C versus LacZ myocytes (P<0.05; in an multiplicity-of-infection–dependent manner), an effect blocked by CaMKII inhibitors KN-93 and autocamtide-2–related inhibitory peptide. This enhanced SR Ca2+ leak may explain reduced SR Ca2+ content, despite measured levels of SR Ca2+-ATPase and Na+/Ca2+ exchange expression and function being unaltered. Ryanodine receptor (RyR) phosphorylation in CaMKII&dgr;C myocytes was increased at both Ser2809 and Ser2815, but FKBP12.6 coimmunoprecipitation with RyR was unaltered. This shows for the first time that acute CaMKII&dgr;C overexpression alters RyR function, leading to enhanced SR Ca2+ leak and reduced SR Ca2+ content but without reducing twitch contraction and Ca2+ transients. We conclude that this is attributable to concomitant enhancement of fractional SR Ca2+ release in CaMKII&dgr;C myocytes (ie, CaMKII-dependent enhancement of RyR Ca2+ sensitivity during diastole and systole) and increased ICa.

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.

Evidence for altered interleukin 18 (IL)-18 pathway in human heart failure

Interleukin (IL)‐18 is the interferon‐γ‐inducing factor and has potent proinflammatory activities. IL‐18 has been recently implicated in atherosclerotic plaque instability and myocardial ischemia‐reperfusion injury. However, it is unknown whether IL‐18 expression is increased in human myocardium or if it has any role in heart failure. We analyzed the expression of IL‐18, its receptor IL‐18Rα, and its endogenous inhibitor, IL‐18 binding protein (IL‐18BP) in myocardial tissue from patients with end‐stage heart failure (ischemic or dilated cardiomyopathy) and controls by use of quantitative real‐time reverse transcriptase polymerase chain reaction, Western blot or immunohistochemical techniques. Plasma levels of IL‐18 were also determined in 48 patients with heart failure. IL‐18 mRNA and protein levels were up‐regulated in the myocardium of patients with ischemic cardiomyopathy. Both ischemic and dilated myocardium showed increased IL‐18Rα levels, suggesting potential biological effects. In addition, mRNA levels of IL‐18 BP were down‐regulated in the failing myocardium. Finally, plasma IL‐18 levels were significantly elevated in patients with heart failure and were higher in those who died at follow‐up than in survivors. The results suggest a potential role for the immunoinflammatory IL‐18 signaling pathway in the pathophysiology of heart failure and identify novel therapeutic targets for future testing.

Effects of Adenovirus-Mediated Sorcin Overexpression on Excitation-Contraction Coupling in Isolated Rabbit Cardiomyocytes

&NA; To evaluate the effect of sorcin on cardiac excitation‐contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad‐sorcin). A &bgr;‐galactosidase adenovirus (Ad‐LacZ) was used as a control. Fractional shortening in response to 1‐Hz field stimulation (at 37°C) was significantly reduced in Ad‐sorcin‐transfected myocytes compared with control myocytes (2.10±0.05% [n=311] versus 2.42±0.06% [n=312], respectively; P<0.001). Action potential duration (at 20°C) was significantly less in the Ad‐sorcin group (458±22 ms, n=11) compared with the control group (520±19 ms, n=10; P<0.05). In voltage‐clamped, fura 2‐loaded myocytes (20°C), a reduced peak‐systolic and end‐diastolic [Ca2+]i was observed after Ad‐sorcin transfection. L‐type Ca2+ current amplitude and time course were unaffected. Caffeine‐induced Ca2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na+‐Ca2+ exchanger (NCX) current revealed a significantly lower SR Ca2+ content and faster Ca2+‐extrusion kinetics in Ad‐sorcin‐transfected cells. Higher NCX activity after Ad‐sorcin transfection was confirmed by measuring the NCX current‐voltage relationship. &bgr;‐Escin‐permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca2+] using a confocal microscope. Under these conditions, caffeine‐mediated SR Ca2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin‐overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca2+‐transient amplitude predominately by lowering SR Ca2+ content via increased NCX activity. The effect of sorcin overexpression on Ca2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation‐contraction coupling. (Circ Res. 2003;93:132‐139.)

Excessive Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase Expression Causes Increased Sarcoplasmic Reticulum Ca2+ Uptake but Decreases Myocyte Shortening

Background—Increasing sarcoplasmic/endoplasmic reticulum (SR) Ca2+-ATPase (SERCA) uptake activity is a promising therapeutic approach for heart failure. We investigated the effects of different levels of SERCA1a expression on contractility and Ca2+ cycling. We tested whether increased SERCA1a expression levels enhance myocyte contractility in a gene-dose–dependent manner. Methods and Results—Rabbit isolated cardiomyocytes were transfected at different multiplicities of infection (MOIs) with adenoviruses encoding SERCA1a (or &bgr;-galactosidase as control). Myocyte relaxation half-time was decreased by 10% (P=0.052) at SERCA1a MOI 10 and by 28% at MOI 50 (P<0.05). Myocyte fractional shortening was increased by 12% at MOI 10 (P<0.05) but surprisingly decreased at MOI 50 (−22%, P<0.05) versus control. SR Ca2+ uptake (in permeabilized myocytes) demonstrated a gene-dose–dependent decrease in Km by 29% and 46% and an increase in Vmax by 37% and 72% at MOI 10 and MOI 50, respectively (all P<0.05 versus control). Ca2+ transient amplitude was increased in Ad-SERCA1a–infected myocytes at MOI 10 (by 121%, P<0.05), but at MOI 50, the Ca2+ transient amplitude was not significantly changed. Caffeine-induced Ca2+ transients indicated significantly increased SR Ca2+ content in Ad-SERCA1a–infected cells, by 72% at MOI 10 and by 87% at MOI 50. Mathematical simulations demonstrate that the functional increase in SR Ca2+-ATPase uptake activity at MOI 50 (and increased cytosolic Ca2+ buffering) is sufficient to curtail the Ca2+ transient amplitude and explain the reduced contraction. Conclusions—Moderate SERCA1a gene transfer and expression improve contractility and Ca2+ cycling. However, higher SERCA1a expression levels can impair myocyte shortening because of higher SERCA activity and Ca2+ buffering.

Over‐expression of FK506‐binding protein FKBP12.6 alters excitation–contraction coupling in adult rabbit cardiomyocytes

This study investigated the function of FK506‐binding protein (FKBP12.6) using adenoviral‐mediated gene transfer to over‐express FKBP12.6 (Ad‐FKBP12.6) in adult rabbit ventricular cardiomyocytes. Infection with a β‐galactosidase‐expressing adenovirus (Ad‐LacZ) was used as a control. Peak‐systolic intracellular [Ca2+] (measured with Fura‐2) was higher in the Ad‐FKBP12.6 group compared to Ad‐LacZ (1 Hz field stimulation at 37°C). The amplitude of caffeine‐induced Ca2+ release was also greater, indicating a higher SR Ca2+ content in the Ad‐FKBP12.6 group. Voltage clamp experiments indicated that FKBP12.6 over‐expression did not change L‐type Ca2+ current amplitude or Ca2+ efflux rates via the Na+–Ca2+ exchanger. Ca2+ transients comparable to those after Ad‐FKBP12.6 transfection could be obtained by enhancing SR Ca2+ content of Ad‐LacZ infected cells with periods of high frequency stimulation. Line‐scan confocal microscopy (Fluo‐3 fluorescence) of intact cardiomyocytes stimulated at 0.5 Hz (20−21°C) revealed a higher degree of synchronicity of SR Ca2+ release and fewer non‐responsive Ca2+ release sites in the Ad‐FKBP12.6 group compared to control. Ca2+ spark morphology was measured in β‐escin‐permeabilized cardiomyocytes at a free [Ca2+]i of 150 nm. The average values of the spark parameters (amplitude, duration, width and frequency) were reduced in the Ad‐FKBP12.6 group. Increasing [Ca2+]i to 400 nm caused coherent propagating Ca2+ waves in the Ad‐FKBP12.6 group but only limited Ca2+ release events were recorded in the control group. These data indicate that FKBP12.6 over‐expression enhances Ca2+ transient amplitude predominately by increasing SR Ca2+ content. Moreover, there is also evidence that FKBP12.6 can enhance the coupling between SR Ca2+ release sites independently of SR content.

Relevance of brain natriuretic peptide in preload-dependent regulation of cardiac sarcoplasmic reticulum Ca2+ ATPase expression.

Background— In heart failure (HF), ventricular myocardium expresses brain natriuretic peptide (BNP). Despite the association of elevated serum levels with poor prognosis, BNP release is considered beneficial because of its antihypertrophic, vasodilating, and diuretic properties. However, there is evidence that BNP-mediated signaling may adversely influence cardiac remodeling, with further impairment of calcium homeostasis. Methods and Results— We studied the effects of BNP on preload-dependent myocardial sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression. In rabbit isolated muscle strips stretched to high preload and shortening isotonically over 6 hours, the SERCA/glyceraldehyde phosphate dehydrogenase mRNA ratio was enhanced by 168% (n=8) compared with unloaded preparations (n=8; P<0.001). Recombinant human BNP at a concentration typically found in end-stage HF patients (350 pg/mL) abolished SERCA upregulation by stretch (n=9; P<0.0001 versus BNP free). Inhibition of cyclic guanosine 3′,5′ monophosphate (cGMP)–phosphodiesterase-5 mimicked this effect, whereas inhibition of cGMP-dependent protein kinase restored preload-dependent SERCA upregulation in the presence of recombinant human BNP. Furthermore, in myocardium from human end-stage HF patients undergoing cardiac transplantation (n=15), BNP expression was inversely correlated with SERCA levels. Moreover, among 23 patients treated with left ventricular assist devices, significant SERCA2a recovery occurred in those downregulating BNP. Conclusions— Our data indicate that preload stimulates SERCA expression. BNP antagonizes this mechanism via guanylyl cyclase-A, cGMP, and cGMP-dependent protein kinase. This novel action of BNP to uncouple preload-dependent SERCA expression may adversely affect contractility in patients with HF.

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.

Overexpression of Integrin β5 Enhances the Paracrine Properties of Circulating Angiogenic Cells via Src Kinase–Mediated Activation of STAT3

Objective—To determine the intracellular mechanisms mediating the angiogenic effects of integrin &agr;v&bgr;5 overexpression in circulating angiogenic cells (CACs). Methods and Results—Integrin &agr;v&bgr;5 is expressed on angiogenic endothelial cells, and integrin &agr;v&bgr;5 activation was shown to improve the reparative functions of endothelial progenitors within the cardiovascular system. CACs were transiently transfected with the full-length cDNA of human integrin &bgr;5 (CAC-ITGB5) or control-vector (CAC-vector). Integrin &bgr;5 overexpression was confirmed using flow cytometry, Western blot, and PCR analysis; it enhanced the angiogenic capacities of CACs in vitro (spheroid and Matrigel angiogenesis assay) and stimulated new vessel formation in vivo (murine hind limb ischemia model). Overexpression of ITGB5 resulted in integrin &agr;v&bgr;5 phosphorylation and activation of Src kinase and signal transducer and activator of transcription (STAT) 3. Furthermore, elevated mRNA and protein expression of the CXC chemokine CXCL8 and the CC chemokine CCL2 was detected in CAC-ITGB5, and conditioned medium from CAC-ITGB5 enhanced the sprouting of coincubated human endothelial cells in a STAT3-, CXCL8-, and CCL2-dependent manner. Conclusion—Src kinase–mediated activation of STAT3 and subsequent angiogenic gene expression mediate the effects of integrin &agr;v&bgr;5 and may be exploited to enhance the paracrine activities of CACs.

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.