Jukka Lehtonen

Cardiac Sarcoidosis and Giant Cell Myocarditis as Causes of Atrioventricular Block in Young and Middle-Aged Adults

Background—Cardiac sarcoidosis (CS) and giant cell myocarditis (GCM) may present as high-degree atrioventricular block (AVB), but their proportion of the causal spectrum of AVB is not well-known. We investigated the prevalence of biopsy-verified CS and GCM in young and middle-aged adults undergoing pacemaker (PM) implantation for AVB. Methods and Results—We used the PM registry of Helsinki University Central Hospital to identify all patients aged 18 to 55 years who underwent PM implantation for AVB between January 1999 and April 2009 and reviewed their medical records. In total, 133 patients had either second- or third-degree AVB as an indication for PM. Of them, 61 had a known cause for AVB, and they were excluded from further analyses. Among the remaining 72 patients with initially unexplained AVB, biopsy-verified CS or GCM was found in 14 (19%) and 4 (6%) patients, respectively. The majority (16/18, 89%) were women. Among the adult patients aged <55 years, the prevalence of CS and GCM combined was 14% (95% CI, 7.7% to 19.3%) of the whole AVB population and 25% (95% CI, 15% to 35%) of those with an initially unexplained AVB. Over an average of 48 months of follow-up, 7 (39%) of 18 patients with CS or GCM versus 1 of the 54 patients in whom AVB remained idiopathic, experienced either cardiac death, cardiac transplantation, ventricular fibrillation, or treated sustained ventricular tachycardia (P<0.001). Conclusions—CS and GCM explain ≥25% of initially unexplained AVB in young and middle-aged adults. These patients are at high risk for adverse cardiac events.

Lipid dynamics and peripheral interactions of proteins with membrane surfaces

A large body of evidence strongly indicates biomembranes to be organized into compositionally and functionally specialized domains, supramolecular assemblies, existing on different time and length scales. For these domains and intimate coupling between their chemical composition, physical state, organization, and functions has been postulated. One important constituent of biomembranes are peripheral proteins whose activity can be controlled by non-covalent binding to lipids. Importantly, the physical chemistry of the lipid interface allows for a rapid and reversible control of peripheral interactions. In this review examples are provided on how membrane lipid (i) composition (i.e., specific lipid structures), (ii) organization, and (iii) physical state can each regulate peripheral binding of proteins to the lipid surface. In addition, a novel and efficient mechanism for the control of the lipid surface association of peripheral proteins by [Ca2+], lipid composition, and phase state is proposed. The phase state is, in turn, also dependent on factors such as temperature, lateral packing, presence of ions, metabolites and drugs. Confining reactions to interfaces allows for facile and cooperative large scale integration and control of metabolic pathways due to mechanisms which are not possible in bulk systems.

Phospholipase A2 as a mechanosensor.

Osmotic swelling of large unilamellar vesicles (LUVs) causes membrane stretching and thus reduces the lateral packing of lipids. This is demonstrated to modulate strongly the catalytic activity of phospholipase A2 (PLA2) toward a fluorescent phospholipid, 1-palmitoyl-2-[(6-pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) residing in LUVs composed of different unsaturated and saturated phosphatidylcholines. The magnitude of the osmotic pressure gradient delta omega required for maximal PLA2 activity as well as the extent of activation depend on the degree of saturation of the membrane phospholipid acyl chains. More specifically, delta omega needed for maximal hydrolytic activity increases in the sequence DOPC < SOPC < DMPC in accordance with the increment in the intensity of chain-chain van der Waals interactions. Previous studies on the hydrolysis of substrate monolayers by C. adamanteus and N. naja PLA2 revealed maximal hydrolytic rates for these two enzymes to be achieved at lipid packing densities corresponding to surface pressures of 12 and 18 mN m-1, respectively. In keeping with the above the magnitudes of delta omega producing maximal activity of Crotalus adamanteus and Naja naja toward PPDPC/DMPC LUVs were 40 and 20 mOsm/kg, respectively. Our findings suggest a novel possibility of regulating the activity of PLA2 and perhaps also other lipid packing density-dependent enzymes in vivo by osmotic forces applied on cellular membranes. Importantly, our results reveal serendipitously that the responsiveness of membranes to osmotic stress is modulated by the acyl chain composition of the lipids.

Lipid microdomains in dimyristoylphosphatidylcholine-ceramide liposomes.

Binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and natural ceramide (up to a mole fraction Xcer = 0.25) were investigated by measuring the excimer:monomer fluorescence emission intensity ratio IE:IM for the pyrene labeled phospholipid probe 1-palmitoyl-2[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC), by monitoring fluorescence polarization of diphenylhexatriene (DPH), as well as using differential scanning calorimetry (DSC). Increasing Xcer > 0.10 both below and above the main transition temperature Tm increased IE:IM for PPDPC maximally approximately 1.6-fold at Xcer = 0.25. Above Tm, and when Xcer approaches 0.10, fluorescence polarization P for DPH increases steeply, reflecting an overall decrease in acyl chain motions. At Xcer = 0.10 there is a discontinuity in P and upon further increase in the content of ceramide a smaller, yet significant increase in P is evident. DSC revealed ceramide to increase in the pretransition temperature until at Xcer exceeding 0.07 this transition was no longer evident. Simultaneously, increasing Xcer up to 0.05 increased Tm from 23.9 to 24.6 degrees C. Total enthalpy delta Hm of the main transition diminished progressively upon increase in Xcer up to approximately 0.10. Above this concentration of ceramide a new endotherm became evident at 22.5 degrees C, and exceeding Xcer = 0.14 this endotherm became dominant. Our results indicate an enrichment of the pyrene labeled phospholipid analog into microdomains concomitant with the formation of a distinct ceramide-enriched phase at Xcer > 0.10.

Cardiac Sarcoidosis: Epidemiology, Characteristics and Outcome over 25 Years in a Nationwide Study

Background— This study was designed to assess the epidemiology, characteristics, and outcome of cardiac sarcoidosis (CS) in Finland. Methods and Results— We identified in retrospect all adult (>18 years of age) patients diagnosed with histologically confirmed CS in Finland between 1988 and 2012. A total of 110 patients (71 women) 51±9 years of age (mean±SD) were found and followed up for outcome events to the end of 2013. The annual detection rate of CS increased >20-fold during the 25-year period, reaching 0.31 in 1×105 adults between 2008 and 2012. The 2012 prevalence of CS was 2.2 in 1×105. Nearly two thirds of patients had clinically isolated CS. Altogether, 102 of the 110 patients received immunosuppressive therapy, and 56 received an intracardiac defibrillator. Left ventricular function was impaired (ejection fraction <50%) in 65 patients (59%) at diagnosis and showed no overall change over 12 months of steroid therapy. During follow-up (median, 6.6 years), 10 patients died of a cardiac cause, 11 patients underwent transplantation, and another 11 patients suffered an aborted sudden cardiac death. The Kaplan–Meier estimates for 1-, 5-, and 10-year transplantation-free cardiac survival were 97%, 90%, and 83%, respectively. Heart failure at presentation predicted poor outcome (log-rank P=0.0001) with a 10-year transplantation-free cardiac survival of only 53%. Conclusions— The detection rate of CS has increased markedly in Finland over the last 25 years. With current therapy, the prognosis of CS appears better than generally considered, but patients presenting with heart failure still have poor long-term outcome.Background— This study was designed to assess the epidemiology, characteristics, and outcome of cardiac sarcoidosis (CS) in Finland. Methods and Results— We identified in retrospect all adult (>18 years of age) patients diagnosed with histologically confirmed CS in Finland between 1988 and 2012. A total of 110 patients (71 women) 51±9 years of age (mean±SD) were found and followed up for outcome events to the end of 2013. The annual detection rate of CS increased >20-fold during the 25-year period, reaching 0.31 in 1×105 adults between 2008 and 2012. The 2012 prevalence of CS was 2.2 in 1×105. Nearly two thirds of patients had clinically isolated CS. Altogether, 102 of the 110 patients received immunosuppressive therapy, and 56 received an intracardiac defibrillator. Left ventricular function was impaired (ejection fraction <50%) in 65 patients (59%) at diagnosis and showed no overall change over 12 months of steroid therapy. During follow-up (median, 6.6 years), 10 patients died of a cardiac cause, 11 patients underwent transplantation, and another 11 patients suffered an aborted sudden cardiac death. The Kaplan–Meier estimates for 1-, 5-, and 10-year transplantation-free cardiac survival were 97%, 90%, and 83%, respectively. Heart failure at presentation predicted poor outcome (log-rank P =0.0001) with a 10-year transplantation-free cardiac survival of only 53%. Conclusions— The detection rate of CS has increased markedly in Finland over the last 25 years. With current therapy, the prognosis of CS appears better than generally considered, but patients presenting with heart failure still have poor long-term outcome. # CLINICAL PERSPECTIVE {#article-title-42}

Diagnosing isolated cardiac sarcoidosis

Abstract.  Kandolin R, Lehtonen J, Graner M, Schildt J, Salmenkivi K, Kivistö SM, Kupari M (Helsinki University Central Hospital, Helsinki, Finland). Diagnosing isolated cardiac sarcoidosis. J Intern Med 2011; 270: 461–468.

Evidence for the formation of microdomains in liquid crystalline large unilamellar vesicles caused by hydrophobic mismatch of the constituent phospholipids.

The excimer-to-monomer fluorescence emission intensity ratio (IE/IM) of the fluorescent probe 1-palmitoyl-2-[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC, 1 mol%) was measured at 30 degrees C as a function of the thickness of fluid liposomal membranes composed of phosphatidylcholines (PCs) with homologous monounsaturated acyl chains of varying lengths N (= number of carbon atoms). Upon decreasing N from di-24:1 PC to di-14:1 PC, the rate of excimer formation was sigmoidally augmented from 0.02 to 0.06. This increase in IE/IM can arise either from enhanced lateral mobility or from the lateral enrichment of PPDPC into domains, or both. Direct evidence for partial lateral segregation of PPDPC being involved is provided by experiments where 1.6 mol% of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N- (5-fluoresceinthiocarbamoyl) (DPPF) was included together with PPDPC into the bilayers. Notably, because of spectral overlap DPPF can function as a resonance energy transfer acceptor for pyrene excimer. Fluorescence intensity ratio (F/Fo) measured at 480 nm for PPDPC/DPPF (yielding F) and PPDPC (yielding Fo) containing membranes as a function of N reveals a sharp maximum for di-20:1 PC, i.e., the quenching of pyrene excimer fluorescence by DPPF is least efficient in this lipid and is enhanced upon either decrease or increase in N. This is compatible with colocalization of DPPF in PPDPC enriched domains when N not equal to 20, whereas in di-20:1 PC these probes appear to be effectively dispersed. The driving force for the enrichment of PPDPC in thin (N < 20) and thick (N > 20) PC matrices is likely to be hydrophobic mismatch of the effective lengths of the matrix phospholipids and the fluorescent probes. We also measured fluorescence polarization (P) for 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as the IE/IM for the intramolecular excimer forming probe 1,2-bis[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) as a function of N. In brief, neither the fluorescence polarization data and nor the measurements of intramolecular chain dynamics using bisPDPC concur with enhanced lateral diffusion as the sole cause for the increase in the IE/IM for PPDPC in thin membranes. Our findings suggest hydrophobic mismatch as the cause of microdomain formation of lipids in fluid, liquid crystalline bilayers, while simultaneously allowing for a high rates of lateral diffusion. Such hydrophobic mismatch-induced compositional fluctuations would also offer one plausible explanation for the chain length diversity observed for biological membranes.

Poly(ethylene glycol)-induced and temperature-dependent phase separation in fluid binary phospholipid membranes.

Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) (PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of the phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large unilamellar liposomes (LUVs). This leads to, in addition to the dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of liposomes. Under these conditions phase separation of the fluorescent lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) embedded in various phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent lipid below Tm was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DMPC, SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of PPDPC cannot be accounted for the 1 degree C increase in Tm for DMPC or for the increase by 0.5 degrees C for DPPC observed in the presence of 20% (w/w) PEG. At a given PEG concentration maximal increase in IE/IM (correlating to the extent of segregation of PPDPC in the different lipid matrices) decreased in the sequence 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) > DPPC > DMPC > SOPC > POPC, whereas no evidence for phase separation in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) LUV was observed (Lehtonen and Kinnunen, 1994, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced dehydration of liposomal membranes provides the driving force for the segregation of the pyrene lipid. In brief, phase separation of PPDPC from the matrix lipid could be attributed to the diminishing effective size of the phosphatidylcholine polar headgroup resulting from its partial dehydration by PEG. This in turn would allow for enhanced van der Waals interactions between the acyl chains of the matrix lipid, which then caused the exclusion of PPDPC due to the perturbing bulky pyrene moiety. Phase separation in DMPC/PPDPC liposomes was abolished by the inclusion of 25 mol % cholesterol and to a lesser extent by epicholesterol.

Diagnosis, Treatment, and Outcome of Giant-Cell Myocarditis in the Era of Combined Immunosuppression

Background— Giant-cell myocarditis often escapes diagnosis until autopsy or transplantation and has defied proper treatment trials for its rarity and deadly behavior. Current therapy rests on multiple-drug immunosuppression but its prognostic influence remains poorly known. We set out to analyze (1) our experience in diagnosing giant-cell myocarditis and (2) the outcome of patients on combined immunosuppression. Methods and Results— We reviewed the histories, diagnostic procedures, details of treatment, and outcome of 32 consecutive patients with histologically verified giant-cell myocarditis treated in our hospital since 1991. Twenty-six patients (81%) were diagnosed by endomyocardial or surgical biopsies and 6 at autopsy or post-transplantation. Twenty-eight (88%) patients underwent endomyocardial biopsy. The sensitivity of transvenous endomyocardial biopsy increased from 68% (19/28 patients) to 93% (26/28) after up to 2 repeat procedures. The 26 biopsy-diagnosed patients were treated with combined immunosuppression (2–4 drugs) including cyclosporine in 20 patients. The Kaplan-Meier estimates of transplant-free survival from symptom onset were 69% at 1 year, 58% at 2 years, and 52% at 5 years. Of the transplant-free survivors, 10/17 (59%) experienced sustained ventricular tachyarrhythmias during follow-up and 3 received intracardiac defibrillator shocks for ventricular tachycardia or fibrillation. Conclusions— Repeat endomyocardial biopsies are frequently needed to diagnose giant-cell myocarditis. On contemporary immunosuppession, two thirds of patients reach a partial clinical remission characterized by freedom from severe heart failure and need of transplantation but continuing proneness to ventricular tachyarrhythmias.

Signaling Mechanism of the AT2 Angiotensin II Receptor: Crosstalk between AT1 and AT2 Receptors in Cell Growth

The peptide angiotensin (Ang) II exerts a range of actions in the cardiovascular, renal, reproductive and central nervous systems. At least two distinct Ang II receptor subtypes have been defined and designated as type 1 (AT1) and type 2 (AT2). The function and signaling mechanism of these receptor subtypes are quite different, and these receptors exert opposite effects on cell growth.