Jason Centracchio

Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome

Long QT syndrome (LQTS) is a heritable disease associated with ECG QT interval prolongation, ventricular tachycardia, and sudden cardiac death in young patients. Among genotyped individuals, mutations in genes encoding repolarizing K+ channels (LQT1:KCNQ1; LQT2:KCNH2) are present in approximately 90% of affected individuals. Expression of pore mutants of the human genes KCNQ1 (KvLQT1-Y315S) and KCNH2 (HERG-G628S) in the rabbit heart produced transgenic rabbits with a long QT phenotype. Prolongations of QT intervals and action potential durations were due to the elimination of IKs and IKr currents in cardiomyocytes. LQT2 rabbits showed a high incidence of spontaneous sudden cardiac death (>50% at 1 year) due to polymorphic ventricular tachycardia. Optical mapping revealed increased spatial dispersion of repolarization underlying the arrhythmias. Both transgenes caused downregulation of the remaining complementary IKr and IKs without affecting the steady state levels of the native polypeptides. Thus, the elimination of 1 repolarizing current was associated with downregulation of the reciprocal repolarizing current rather than with the compensatory upregulation observed previously in LQTS mouse models. This suggests that mutant KvLQT1 and HERG interacted with the reciprocal wild-type alpha subunits of rabbit ERG and KvLQT1, respectively. These results have implications for understanding the nature and heterogeneity of cardiac arrhythmias and sudden cardiac death.

Hepatocyte Growth Factor Modulates Matrix Metalloproteinases and Plasminogen Activator/Plasmin Proteolytic Pathways in Progressive Renal Interstitial Fibrosis

Evidence suggests that hepatocyte growth factor (HGF) ameliorates renal fibrosis in animal models of chronic renal disease by promoting extracellular matrix catabolism. This study examined the molecular mechanisms of HGF-induced alterations in matrix degradation both in vitro and in vivo. In vitro, HGF increased the collagen catabolizing activity of human proximal tubular epithelial cells (HKC) that were treated with TGF-beta1. Increased collagen catabolism was associated with enhanced activity of both matrix metalloproteinases (MMP) and plasminogen activators (PA)/plasmin proteolytic pathways. HGF abrogated TGF-beta1-induced production of the profibrotic tissue inhibitor of metalloproteinase-2 (TIMP-2) and plasminogen activator inhibitor-1 (PAI-1). In addition, HGF induced the production of MMP-9. In vivo, continuous infusion of HGF in the rat remnant kidney model ameliorated renal fibrosis and tubulointerstitial collagen deposition. This was associated with increased tubular expression of MMP-9, enhanced in situ gelatinolytic activity, partially restored plasmin activity and decreased expression of TIMP-2 and PAI-1 in tubular cells, and upregulation of renal TIMP-3 expression. Conversely, blocking of endogenous HGF by an anti-HGF neutralizing antibody increased renal fibrosis and interstitial collagen. This was accompanied by decreased tubular expression of MMP-9, less in situ proteolytic activity, and elevated expression of TIMP-2 and PAI-1 in tubular cells. Collectively, these findings demonstrate that HGF ameliorates renal fibrosis by enhancing extracellular matrix catabolism via both MMP and the PA/plasmin proteolytic pathways.

Estradiol promotes sudden cardiac death in transgenic long QT type 2 rabbits while progesterone is protective

BACKGROUND Postpubertal women with inherited long QT syndrome type 2 (LQT2) are at increased risk for polymorphic ventricular tachycardia (pVT) and sudden cardiac death (SCD), particularly during the postpartum period. OBJECTIVE To investigate whether sex hormones directly modulate the arrhythmogenic risk in LQTS. METHODS Prepubertal ovariectomized transgenic LQT2 rabbits were treated with estradiol (EST), progesterone (PROG), dihydrotestosterone (DHT), or placebo (OVX). RESULTS During 8 weeks of treatment, major cardiac events-spontaneous pVT or SCD-occurred in 5 of the 7 EST rabbits and in 2 of the 9 OVX rabbits (P <.05); in contrast, no events occurred in 9 PROG rabbits and 6 DHT rabbits (P <.01 vs PROG; P <.05 vs DHT). Moreover, EST increased the incidence of pVT (P <.05 vs OVX), while PROG reduced premature ventricular contractions, bigeminy, couplets, triplets, and pVT (P <.01 vs OVX; P <.001 vs EST). In vivo electrocardiographic monitoring, in vivo electrophysiological studies, and ex vivo optical mapping studies revealed that EST promoted SCD by steepening the QT/RR slope (P <.05), by prolonging cardiac refractoriness (P <.05), and by altering the spatial pattern of action potential duration dispersion. Isoproterenol-induced Ca(2+) oscillations resulted in early afterdepolarizations in EST-treated hearts (4 of 4), while PROG prevented SCD by eliminating this early afterdepolarization formation in 4 of the 7 hearts (P = .058 vs EST; P <.05 vs OVX). Analyses of ion currents demonstrated that EST increased the density of I(Ca,L) as compared with OVX (P <.05) while PROG decreased it (P <.05). CONCLUSION This study reveals the proarrhythmic effect of EST and the antiarrhythmic effect of PROG in LQT2 in vivo, outlining a new potential antiarrhythmic therapy for LQTS.

Redox modification of ryanodine receptors by mitochondria-derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts

•  Ageing is associated with increased risk of sudden cardiac death due to malignant arrhythmias. •  Shortened refractoriness of Ca2+ release due to increased activity of Ca2+ release channels (RyRs) is recognized as an important contributor to cardiac‐triggered arrhythmias. However, molecular mechanisms of RyR dysfunction and its contribution to arrhythmias in ageing remain to be examined. •  Using ventricular myocytes isolated from old rabbit hearts we demonstrate that age‐associated increase in rate of production of reactive oxygen species (ROS) by mitochondria leads to the thiol‐oxidation of RyRs, which underlies the hyperactivity of the channels and thus shortened refractoriness of Ca2+ release in cardiomyocytes from the ageing heart. Mitochondria‐specific scavenging of ROS in old myocytes restored the redox status of RyRs, reducing SR Ca2+ leak and arrhythmogenic spontaneous Ca2+ waves. •  We conclude that increased ROS production by mitochondria contributes to age‐associated increased risk of stress‐induced arrhythmia and sudden cardiac death through thiol‐modifications of RyRs.

Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines

We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K(+) channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K(+) current (I(Ks) and I(Kr), respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, I(Ks) and I(Kr), without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated I(Kr) in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in I(Kr) upon KvLQT1 expression. Deletion of the KvLQT1 NH(2)-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced I(Ks) in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.

Expression of Na+/H+ Exchanger Isoforms in Inner Segment of Inner Medullary Collecting Duct (IMCD3)

Abstract. Na+/H+ exchangers (NHE) play a critical role in many cellular and transport processes in the inner medullary collecting duct (IMCD). Morphologically, the IMCD is divided into the outer (IMCD1), middle (IMCD2), and inner (IMCD3) segments. The inner, IMCD3 segment contains only one cell type, the IMCD cell, which is distinct in ultrastructure and in function from the principal and intercalated cells that are present in other portions of the IMCD. NHEs constitute a gene family containing several isoforms (NHE1, NHE2, NHE3, NHE4 and NHE5) which possess distinct characteristics and serve specialized functions. To understand the molecular basis of NHE-related processes in the IMCD, it is critical to know the molecular identity of the NHEs in this tubule segment. The purpose of the present study was to identify the NHE isoforms present and their polar distribution in IMCD3. Applying the reverse transcription-polymerase chain reaction (RT-PCR) technique to IMCD3 (obtained from distal 50% of inner medulla) of mouse and rat kidneys, we found that NHE1, NHE2 and NHE4, but not NHE3 were expressed in both species. The polar localization of NHE in IMCD3 was examined in tubules isolated from rats and perfused in vitro with HEPES-buffered solutions under isotonic conditions. pHi was measured by BCECF fluorescence. Na+-dependent, amiloride-inhibitable pHi recovery from cell acidification (consistent with NHE) was detected in the basolateral, but not the apical, membrane of IMCD3. We conclude that NHE1, NHE2 and NHE4, but not NHE3, are present in both the mouse and rat IMCD3. Functionally, NHE is limited to the basolateral membrane. Additional studies are needed to determine the physiological roles and regulation of basolateral NHE isoforms in this tubule segment.