The research in our laboratory is focused on elucidated the molecular mechanisms of cardiac arrhythmogenesis and conduction disease. We are using genetically engineered mouse models of human arrhythmogenic disorders to investigate arrhythmia mechanisms at the level of the whole heart, cardiomyocyte and molecule. This integrative approach is accomplished by using the murine in vivo electrophysiology technique in combination with the patch clamp technique and standard molecular and biochemical techniques. To investigate arrhythmogenesis at the level of the whole heart, we insert a 2-french octapolar electrophysiology catheter into the right internal jugular vein of an anesthetized mouse with the distal 4 poles in the right ventricle and the proximal 4 poles in the right atrium. We can then stimulate and record electrograms from either cardiac chamber individually to perform programmed stimulation and assess the presence of arrhythmia inducibility and conduction system disease, just as in humans. If specific disorders of arrhythmogenesis are observed, the underlying voltage-dependent ionic currents are investigated at the cellular level using the patch clamp technique. Individual cardiac myocytes are dissociated from the ventricle and atrium and assayed using the whole-cell configuration of the patch clamp technique. By employing both the voltage-clamp and current-clamp modes of the patch clamp technique, information can be obtained about abnormalities in the expression and kinetics of the underlying voltage-dependent currents that may be related to the genetic mutation and be proarrhythmic. Based upon which ion channels appear to be modulated by the engineered mutation, and if they correlate with any induced arrhythmias or conduction disorders, we can assess if the induced mutation affects channel protein and message expression using immunoblot and quantitative RT-PCR, respectively. In addition, alterations in channel distribution are detected using immunocytochemistry and in situ hybridization, in conjunction with our core histology laboratory. Finally, pull down assays can help to identify if binding occurs between modulated ion channels and mutant enzymes and if so, which domains are involved in these interactions. Employing this stepwise approach, the arrhythmogenic effect of mutations in specific transcription factors, enzymes or channels can be elucidated and correlated at the levels of the whole heart, cardiomyocyte and molecule.
|Figure 1. Telemetry ECG recordings of the onset of spontaneous polymorphic ventricular tachycardia/ventricular fibrillation recorded from two different N-cadherin CKO mice. The left-hand portion of the panel shows normal sinus rhythm with the onset of arrhythmia at arrows #1 and #3.|
Currently, we are investigating the mechanisms underlying sudden death in a mouse model of non-ischemic dilated cardiomyopathy. Cardiac specific deletion of the adherence junction protein N-cadherin induces dilated cardiomyopathy and spontaneous ventricular fibrillation with loss of the intercalated disks. While deletion of N-cadherin is expected to lead to loss of adherence junctions in the heart, since N-cadherin is the only classic cadherin expressed in the mammalian heart, it was surprising that it also lead to down-regulation of connexin43 with loss of the gap junctions. Therefore, it appears that the adherence junctions stabilize the gap junctions, either directly or indirectly, which leads to slowing in the epicardial conduction velocity, an increase in the anisotropy ratio and arrhythmogenesis. Investigations are presently under way to determine the mechanism by which N-cadherin stabilizes connexin in the heart as this has important implications for the therapy and treatment of sudden death in humans which claims ~500,000 lives annually in the United States.
|Figure 2. Immunocytochemical staining again N-cadherin and connexin43 shows normal levels of both proteins in the ventricle of wild-type control animals (A & B) while there is loss of N-cadherin and connexin43 in the ventricular myocardium of N-cadherin CKO mice (C & D).|
We are also investigating factors involved with maintenance and function of the cardiac conduction system. Specifically, we have shown that expression of the homeodomain-only protein (Hop) is confined predominantly to the adult, murine cardiac conduction system and that deletion of the Hop locus leads to infra-nodal conduction defects.
|Figure 3. Surface ECG recordings and intracardiac electrograms from a Hop null mouse and its wild-type littermate. Note the increased QRS duration and HV-interval (arrows) in the absence of Hop in the mature mouse heart.|
In the mature heart Hop does not appear necessary for normal patterning of the cardiac conduction system but conduction defects seem to be due to loss of connexin40 which is the major gap junction protein in the murine conduction system.
|Figure 4. Serial frozen sections of newborn Hop+/- (A through C) and Hop-/- (D through F) hearts were stained for β-galactosidase activity (A and D) and connexin40 (B and C, E and F). Areas of the CCS are outlined. Although there was strong expression of connexin40 seen in the Hop+/- CCS (B), the Hop-/- CCS showed expression of connexin40 in a much smaller area that did not extend beyond the proximal AVN and His-bundle (E).|
We are further investigating the role of Hop in conduction system disease to determine its effects upon the expression and function of voltage-dependent ion channels and intracellular signaling in the mature, mammalian conduction system.
Lenschow, D.H., V. Patel and A. Isbell. Measurements of fine-scale structure at the top of marine stratocumulus. Bull. Amer. Meteor. Soc. 69:891-894, 1988.
Martin, A.R., V.V. Patel, L. Faille and A. Mallart. Presynaptic calcium currents recorded from calyciform nerve terminals in the lizard ciliary ganglion. Neurosci. Lett. 105:14-18, 1989.
Patel, V.V., J.H. Caldwell and S.R. Levinson. Cellular surface charge influences the function of the rskm1 Na+ channel. Neuron. 17:1002-1008, 1994.
Patel, V.V. and S.R. Levinson. Surface charge screening and block of the Na+ channel by Ca2+ occurs at two independent sites. Biophys. J. 70:2370-2375, 1995.
Fox, J.C. and V.V. Patel. Apoptosis and the cardiovascular system. ACC Curr. J. Rev. 7:13-15, 1998.
Marchlinski, F.E., E.S. Zado, D.J. Callans, V.V. Patel, M.S. Ashar, H.H. Hsia and A.M. Russo. Hybrid therapy for ventricular arrhythmia management. Cardiol. Clin. 18:391-406, 2000.
St. John Sutton, M. and V.V. Patel. Right ventricular dysplasia. In: Weigers, S.E, Plappert, T. and St. John Sutton, M. (eds): Echocardiography in Practice: A Case-Oriented Approach. Martin Dunitz, Ltd. 2001: 96-99.
Patel, V.V., H. Nayak and F.E. Marchlinski. Interpretation and application of stored electrograms. Clin. Electrophys. Rev. 5:96-103, 2001
Pavri, B.B., R.T. Ho, V.V. Patel, D.J. Callans, S. Folley and D.Z. Kocovic. Unipolar Defibrillator? Pacing Clin. Electrophysiol. 24:244-246, 2001.
Patel, V.V., V.A. Ferrari, N. Narula, S.E. Weigers and M.G. St. John-Sutton. Right ventricular dysplasia in an asymptomatic young man: an uncommon case with biventricular involvement and no known familial history. J. Amer. Soc. Echocard. 14:317-320, 2001.
Rho, R.W., V.V. Patel and D.Z. Kocovic. Current concepts of biventricular pacing in heart failure. In: Jessup M (ed): Heart Failure: A Clinicians Guide to Ambulatory Diagnosis and Treatment. Humana Press. 2002: 113-124.
Callans, D.J., F-J Ren, N. Narula, V. Patel, J. Michele, A. Gelzer and S.M. Dillon. (2002). Left ventricular catheter ablation using direct, intramural ethanol injection in swine. J. Interven. Card. Electrophysiol. 6:225-231, 2002.
Patel, V.V., J-F Ren and F.E. Marchlinski. A comparison of left atrial size by transthoracic echocardiography and magnetic catheter mapping. Pacing Clin. Electrophys. 25:14-17, 2002.
Patel, V.V., F-J Ren, M.E. Jeffery, T.J. Plappert, M.G. St. John Sutton and F.E. Marchlinski. A comparison of left atrial volume assessed by magnetic endocardial catheter mapping versus transthoracic echocardiography. Am. J. Cardiol. 91:351-354, 2003.
Rho, R.W., V.V. Patel, E.P. Gerstenfeld, S. Dixit, J.W. Poku, H.M. Ross, D.J. Callans and D.Z. Kocovic. Elevations in ventricular pacing threshold with the use of the Y adaptor: implications for biventricular pacing. Pacing Clin. Electrophysiol. 26:747-751, 2003.
Dixit, S., E.P. Gerstenfeld, R.W. Rho, V. Patel, D.J. Callans and F.E. Marchlinski. Change in distant atrial activation patterns during circumferential pacemapping of pulmonic vein ostium: implications for localizing triggers for atrial fibrillation.J. Interv. Card. Electrophysiol. 8:187-194, 2003.
Arad, M., I.P. Moskowitz, V.V. Patel, F. Ahmad, A.R. Perez-Atayde, D.B. Sawyer, M. Walter, G.H. Li, P.G. Burgon, C.T. Magurie, D. Stapleton, J.P. Schmidt, X.X. Guo, A. Pizard, S. Kupershmidt, D.M. Roden, C.I. Berul, C.E. Seidman and J.G. Seidman. Transgenic mice overexpressing mutant PRKAG2 defines the cause of Wolff-Parkinson-White syndrome in glycogen storage cardiomyopathy. Circulation. 107:2850-2856, 2003.
Patel, V.V., M. Arad, I.P.G. Moskowitz, C.T. Maguire, D. Branco, J.G. Seidman, C.E. Seidman and C.I. Berul. Electrophysiological characterization and postnatal development of ventricular preexcitation in a mouse model of cardiac hypertrophy and Wolff-Parkinson-White syndrome. J. Am. Coll. Cardiol. 42:942-951, 2003.
Maguire, C.T., H. Wakimoto, V.V. Patel, P.E. Hammer and C.I. Berul. Implications of ventricular arrhythmia vulnerability during murine electrophysiology studies.Physiol. Genomics. 15:84-91, 2003.
Rajawat, Y., E.P. Gerstenfeld, V.V. Patel, R.W. Rho, S. Dixit, D.J. Callans and F.E. Marchlinski. Surface ECG criteria for localizing the pulmonary vein origin of spontaneous atrial complexes: validation using intracardiac recordings.Pacing Clin. Electrophysiol. 2004; 27:182-188.
Moskowitz, I.P.G., A. Pizard, V.V. Patel, B.G. Bruneau, J.B. Kim, S. Kupershmidt, D. Roden, C.I. Berul, C.E. Seidman, J.G. Seidman. The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development 131:4107-4116, 2004.
Patel, V.V., R.W. Rho, E.P. Gerstenfeld, H.H. Hsia, D.J. Callans, F.E. Marchlinski. Right bundle branch block ventricular tachycardias: septal versus lateral ventricular origin based on activation time to the right ventricular apex. Circulation 110:2582-2587, 2004.
Rajawat, Y., V.V. Patel, E.P. Gerstenfeld, H. Nayak, F.E. Marchlinski. Advantages and pitfalls of combining device-based and pharmacological therapies for the treatment of ventricular arrhythmias: experience from a large tertiary center. Pacing Clinical Electrophysiology 27:1670-1681, 2004.
Kostetskii, I., J. Li, Y. Xiong, R. Zhou, V.A. Ferrari, V.V. Patel, J.D. Molkentin, G.L. Radice. Induced deletion of the N-cadherin gene in the heart leads to dissolution of the intercalated disc structure. Circulation Research 96:346-54, 2005.
Russo, A.M., W. Sauer. E.P. Gerstenfeld, H.H. Hsia, D. Lin, J.M. Cooper, S. Dixit, R.J. Verdino, H.M. Nayak, D.J. Callans, V. Patel, F.E. Marchlinski. Defibrillation threshold testing: is it really necessary at the time of implantable cardioverter-defibrillator insertion? Heart Rhythm 2:456-61, 2005.
Ismat, F.A., Z. Maozhen, H. Kook, B. Huang, R. Zhou, V.A. Ferrari, J.A. Epstein, V.V. Patel. The homeobox protein Hop functions in the adult cardiac conduction system. Circulation Research 96:898-903, 2005.
Li, J.*, V.V. Patel*, I. Kostetskii, Y. Xiong, A.F. Chu, J.T. Jacobson, C. Yu, G.E. Morley, J.D. Molkentin, G.L. Radice. Cardiac-specific loss of N-cadherin leads to alteration in connexins with conduction slowing and arrhythmogenesis. Circulation Research 97:474-481, 2005. [Cover].
Li, J., V.V Patel, G.L. Radice. Dysregulation of cell adhesion molecules and cardiac arrhythmogenesis. Clin Med Research, 4:42-52, 2006.
Liu, F., F.A. Ismat, V.V. Patel. Role of homeodomain-only protein in the cardiac conduction system. Trends Cardiovasc Med. 2006;16:193-198.
- Murine in vivo EPS Protocol
- Murine EPS Drug Doses
- Procedure for Adult Mouse Myocyte Isolation
- Solutions for Adult Mouse Myocyte Isolation
Vickas Patel, M.D., Ph.D.
Assistant Professor of Medicine
Molecular Cardiology Research Center and
Section of Cardiac Electrophysiology
The Cardiovascular Institute
University of Pennsylvania
907 BRB II/III
421 Curie Blvd.
Philadelphia, PA 19104
Office: (215) 898-2800
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