Proc. Natl. Acad. Sci. USA Vol. 94, pp. 5444–5449, May 1997 Physiology Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function MARGARET V. WESTFALL*, ELIZABETH M. RUST, AND JOSEPH M. METZGER Department of Physiology, School of Medicine, University of Michigan, Ann Arbor, MI, 48109-0622 Communicated by James A. Spudich, Stanford University, Stanford, CA, March 7, 1997 (received for review November 5, 1996) ABSTRACT The functional significance of the develop- changes remains unclear, because developmental transitions in mental transition from slow skeletal troponin I (ssTnI) to other contractile protein isoforms occur over the same time cardiac TnI (cTnI) isoform expression in cardiac myocytes interval as the TnI isoform transition. remains unclear. We show here the effects of adenovirus- TnI isoforms are also postulated to influence cardiac myo- mediated ssTnI gene transfer on myofilament structure and filament pH sensitivity (4, 5). Contractile function decreases function in adult cardiac myocytes in primary culture. Gene markedly during acute myocardial ischemia (4), and acidosis transfer resulted in the rapid, uniform, and nearly complete plays a significant role in this decreased function by reducing replacement of endogenous cTnI with the ssTnI isoform with myofilament Ca21 sensitivity (4–7). Solution studies indicate no detected changes in sarcomeric ultrastructure, or in the that TnI may play a role in this phenomenon because acidic isoforms and stoichiometry of other myofilament proteins pH-induced decreases in Ca21 binding to andyor subsequent compared with control myocytes over 7 days in primary conformational changes within troponin C (TnC) are in- culture. In functional studies on permeabilized single cardiac creased in magnitude in the presence of TnI (8). Studies using myocytes, the threshold for Ca21-activated contraction was permeabilized muscle preparations also have implicated TnI in significantly lowered in adult cardiac myocytes expressing this pH effect. For example, the acidosis-induced shift in ssTnI relative to control values. The tension–Ca21 relation- myofilament Ca21 sensitivity is greater in cTnI-expressing ship was unchanged from controls in primary cultures of adult myocardium than in slow skeletal muscle fibers (9, 10) or cardiac myocytes treated with adenovirus containing the adult fetalyneonatal myocardium (3, 5), which express ssTnI (1, 2). cardiac troponin T (TnT) or cTnI cDNAs. These results Together, these results suggest that TnI influences the myo- indicate that changes in Ca21 activation of tension in ssTnI- filament Ca21 sensitivity response to acidosis in an isoform- expressing cardiac myocytes were isoform-specific, and not dependent manner. However, direct confirmation that TnI due to nonspecific functional changes resulting from overex- isoforms influence the acidosis-mediated shift in Ca21 sensi- pression of a myofilament protein. Further, Ca21-activated tivity is difficult to assess in cardiac myocytes because other tension development was enhanced in cardiac myocytes ex- contractile protein isoforms also differ across muscle lineages pressing ssTnI compared with control values under conditions and during myocardial development (11). There are no pre- mimicking the acidosis found during myocardial ischemia. vious reports documenting the effect of ssTnI alone on Ca21- These results show that ssTnI enhances contractile sensitivity activated tension in adult cardiac myocytes. to Ca21 activation under physiological and acidic pH condi- Thus, the major goal of the present study is to define the tions in adult rat cardiac myocytes, and demonstrate the functional role of ectopically expressed ssTnI in adult cardiac utility of adenovirus vectors for rapid and efficient genetic myocytes as a means of further understanding the function of modification of the cardiac myofilament for structurey TnI isoforms within the contractile apparatus at physiological function studies in cardiac myocytes. and acidic pH. Recombinant adenovirus is used to deliver a ssTnI expression cassette into adult cardiac myocytes in pri- The thin filament protein troponin I (TnI) plays an essential mary culture. Western blot and immunohistochemistry anal- role in the regulation of striated muscle contraction. One yses of cultured myocytes show rapid and efficient expression approach to understanding the role of TnI within the contrac- and incorporation of ssTnI into the myofilament. Further- tile apparatus is to study functional differences among TnI more, ectopic ssTnI expression occurs without any detectable isoforms. TnI isoforms are expressed in a muscle lineage- effects on the isoform composition or stoichiometry of other specific and developmentally regulated pattern (1, 2). In contractile assembly proteins, or on the highly ordered struc- cardiac muscle, there are two developmentally regulated TnI ture of the contractile lattice. Importantly, functional mea- isoforms. The slow skeletal TnI (ssTnI) isoform is expressed in surements of Ca21-activated tension under physiological- fetal myocardium and the cardiac TnI (cTnI) isoform is activating conditions directly demonstrate, for the first time to expressed exclusively in adult myocardium (1, 2). During our knowledge, that TnI isoforms influence adult cardiac cardiac development, there are also marked alterations in myocyte contractile function. Specifically, we observe a lower cardiac contractile function, including an increase in the threshold for Ca21 activation in adult cardiac myocytes ex- steepness and a rightward shift in the tension-Ca21 relation- pressing ssTnI, a phenotype similar to that of fetalyneonatal ship (1, 3). Thus, there is a correlation between TnI isoform myocardium (1, 3). Functional studies further show that pH expression and changes in myofilament function. However, the sensitivity is reduced in the adult cardiac myocytes expressing precise role of TnI isoforms in mediating these functional Abbreviations: CMV, cytomegalovirus; cTnI, cardiac troponin I; LC1, The publication costs of this article were defrayed in part by page charge myosin light chain 1; LC2, myosin light chain 2; mu, map unit; nH, Hill 21 payment. This article must therefore be hereby marked ‘‘advertisement’’ in coefficient; pCa, 2log[Ca ]; pCa50, pCa required to produce 50% of accordance with 18 U.S.C. §1734 solely to indicate this fact. maximal tension; ssTnI, slow skeletal troponin I; TnC, troponin C; TnI, troponin I; TnT, troponin T; Tm, tropomyosin. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA *To whom reprint requests should be addressed at: Department of 0027-8424y97y945444-6$2.00y0 Physiology, University of Michigan, 1301 East Catherine St., 7730 PNAS is available online at http:yywww.pnas.org. Medical Science II, Ann Arbor, MI 48109-0622. 5444 Downloaded by guest on September 29, 2021 Physiology: Westfall et al. Proc. Natl. Acad. Sci. USA 94 (1997) 5445 ssTnI, indicating that TnI isoform expression significantly mgyml; Sigma) for 15 min. After increasing the [Ca21]inthe influences the myofilament response to acidosis. digestion solution (1 mM), and continuing the perfusion for 15 min, isolated ventricles were minced into pieces and gently METHODS shaken in digestion solution with occasional trituration using silanized pasteur pipets. Undigested ventricular tissue was Generation of Adenoviral Vectors. To construct recombi- removed using a 230-mm mesh sieve. The cell suspension was nant adenovirus vectors, the plasmids pAdCMVssTnI and centrifuged, and the [Ca21] was increased to 1.75 mM after pJM17 (12) were cotransfected by calcium phosphate into a resuspending pelleted cells in KHB-A containing 2% BSA. HEK 293 cell line (Fig. 1A). The shuttle plasmid pAdCM- After centrifuging cells again, they were resuspended in culture VssTnI contained adenovirus serotype 5 sequences 0–1 map media containing serum [DMEM containing 50 unitsyml units (mu) and 9–16 mu flanking an expression cassette penicillin plus 50 mgyml streptomycin (PyS), and 5% fetal containing the cytomegalovirus (CMV) promoter, the coding bovine serum]. Rod-shaped ventricular myocytes were plated sequence for the full-length rat ssTnI cDNA and its 39 un- onto laminin-coated coverslips in DMEM1PyS1fetal bovine translated region (13), and the simian virus 40 polyadenylyla- serum for 2 hr and then infected with recombinant adenovirus tion signal. The pJM17 plasmid was a 0–100 mu derivative of ('170 plaque-forming unitsycell) in serum-free media adenovirus serotype 5 containing a partial deletion in the E3 (DMEM1PyS). Serum-free medium (2 ml) was added 1 hr region and a 4.3-kb pBRX insert at 3.7 mu. The insert allowed later and changed every 2–3 days thereafter. replication of the plasmid in bacteria but made the viral Analysis of Protein Composition by Gel Electrophoresis and genome too large to be packaged into the virus capsid (12). Immunoblotting. Myofilament proteins were analyzed by col- After homologous recombination, the expression cassette in lecting 10 to 20 ventricular myocytes on a glass micropipet tip pAdCMVssTnI replaced the pBRX insert and the E1 region of and then transferring the myocytes to microcentrifuge tubes the genome (1–9 mu) in pJM17, thus making the recombinant containing 10 ml of sample buffer for separation by gel adenovirus, AdCMVssTnI, capable of being packaged but electrophoresis as described previously (17). For Western blot replication defective. The HEK 293 cell line is adenovirus