Chief, Section on Cell Structure and Function
Laboratory of Cell Biology
Department of Cell and Molecular Biology
Phone: 213-353-7030
Fax: 213-273-8088
Email: fkalinec@hei.org
Academic Affiliations
Department of Otolaryngology
University of Southern California, School of Medicine
Department of Cell and Neurobiology
University of Southern California, School of Medicine
Education
- Universidad Nacional de Córdoba, Argentina Ph.D. (Biophysics) 1989
- Universidad Nacional de Córdoba, Argentina, Physicist 1976
Professional Experience
Visiting Associate, NIDCD-NIH, Bethesda, MD, USA - 1995-1996
Visiting Fellow, NIDCD-NIH, Bethesda, MD, USA - 1990-1994
Assistant Professor of Physics, School of Sciences, Universidad de Cordoba, Cordoba, Argentina - 1986-1990.
Research and Selected Publications
I. Cytoskeletal regulation of outer hair cell motility
Introduction
The human ear is capable of both amplifying faint sounds to increase our hearing range and reducing the energy of loud sounds to protect our ears from damage. It can do that because of the “cochlear amplifier,” an active mechanism that increases the sensitivity and frequency discrimination of the inner ear. Damage of the cochlear amplifier by, for example, acoustic trauma, ototoxic drugs or aging, causes sensorineural hearing loss.
The mechanism of cochlear amplification is based in the motile properties of auditory sensory cells known as outer hair cells (OHCs). When stimulated, OHCs change their length either over a period of seconds (slow motility) or in the microsecond range (fast motility). OHC slow motility can modify the mechanics of the organ of Corti changing the distance between the tectorial and basilar membranes. OHC fast motility, in turn, works at the same frequency of the sound, increasing up to 100 times the peak amplitude of the basilar membrane movement and, consequently, cochlear sensitivity. The motor mechanism responsible for their unique electromotile response is hold in the plasma membrane of outer hair cells.
a – Involvement of Rho GTPases in the regulation of OHC motility
Results from our laboratory suggested that the gain of the cochlear amplifier is regulated via changes in OHC motility associated with dynamic reorganization of the cell skeleton (cytoskeleton) mediated by small GTPases of the RHO family (Kalinec et al, 2000). Our experiments showed, for instance, that inhibition of the small GTPase RhoA and its downstream target, the Rho-kinase ROCK, decrease OHC electromotile amplitude, a parameter directly associated with cochlear amplification. We used the term “mechanical homeostasis” for the process of constant adjustment of structural parameters that automatically bring OHCs near the optimal working point for each condition. A self-regulated homeostatic response, however, may be insufficient to protect the cochlea from sudden bursts of high-energy noise. We also found that the neurotransmitter acetylcholine (ACh), released by the efferent terminals innervating OHCs, is able to disengage the “automatic gain control” of the cochlear amplifier and rapidly inhibit amplification. This effect could be important when the auditory input reaches levels that might induce cochlear damage. Our experiments suggested that ACh influences OHC electromotility by activating a signaling pathway mediated by RhoA but not by ROCK. Thus, these results suggest that OHC motility is regulated via ROCK-dependent and ROCK-independent pathways (Zhang et al, 2002; Zhang et al, 2003). Although these results strongly implicate the involvement of the cytoskeleton in the regulation of the OHC motile response, the precise molecular mechanisms underlying this regulatory process is still unknown. Recent studies in our laboratory indicated that the dynamic control of the process of polymerization and depolymerization of actin filaments —via the Rho/ROCK/LIMK/Cofilin-mediated signaling pathway— is also critical for the OHC motile response. These results extend our understanding of a basic mechanism of both normal human hearing and deafness and revealing the key role of the cytoskeleton in the regulation of outer hair cell electromotility.
b - Prestin-dependent and prestin-independent motility of cochlear outer hair cells
Classically, OHC motility has been divided in two types of responses: fast and slow. Fast motility, also known as electromotility, is voltage-dependent and follows, cycle-by-cycle, changes in membrane potential at auditory frequencies and beyond. Electromotility has been associated with both conformational changes and rearrangement of the voltage-sensitive protein “prestin”, which is embedded in the OHC’s external (plasma) membrane. The term ‘‘slow motility’’ is commonly used to describe changes in OHC shape taking place over periods of milliseconds to seconds and even minutes. It has been proposed that slow motility modulates fast motility and contributes to the fine-tuning of the acoustic transduction process. The cellular and molecular mechanisms involved in OHC slow motility, however, are not completely understood yet, and the criteria to distinguish between fast and slow motility is still based on the speed of the response.
We have developed a novel experimental approach that uses of a computerized method for the continuous analysis of OHC motility based on the "Dynamic Image Analysis System” software (DIAS. Soll Technologies Inc., Iowa City, IA). With this software, digitally recorded images of isolated OHCs may be thoroughly analyzed, providing automatically frame-by-frame values of length, diameter, longitudinal section area and other parameters of the recorded cells. By performing careful measurements in OHCs exposed to different stimuli, we first demonstrated that some responses commonly identified as “slow OHC motility” were actually the result of two different mechanisms, one mediated by prestin, the membrane-embedded motor molecule, and other prestin-independent (Matsumoto & Kalinec, 2005a). Importantly, we found that electrical stimulation evokes significant prestin-dependent changes in the length, width and area of the longitudinal section of OHCs, but not in their volume. In contrast, prestin-independent responses elicited at constant membrane potential are associated with changes in cell length, width and volume without significant changes in their longitudinal section area (Matsumoto & Kalinec, 2005a)..
Next, we described a novel analytical technique, based on a simple theoretical model and continuous measurement of changes in cell length and longitudinal section area, to evaluate the contribution of each one of these mechanisms to the motile response of OHCs. We demonstrated that, if the relative change in OHC length (L) during the motile response is expressed as L = A2 x V-1 (with A and V being the relative changes in longitudinal section area and volume, respectively), A2 will describe the contribution of the prestin-dependent while V-1 will describe the contribution of the prestin-independent mechanisms (Matsumoto & Kalinec, 2005b). Thus, measurements of the relative changes in any two of these cellular morphological parameters (L, A or V) would be necessary and sufficient for characterizing any OHC motile response. This simple approach provides access to information previously unavailable, and may become a novel and important tool for increasing our understanding of the cellular and molecular mechanisms of OHC motility (Matsumoto & Kalinec, 2005b).
Thus, we have now a novel and powerful tool for investigating the molecular mechanisms underlying the generation and regulation of the prestin-dependent and prestin-independent OHC motile responses.
c - Molecular organization of the outer hair cell plasma membrane
The basal and lateral regions of the plasma membrane of cochlear outer hair cells are structurally and functionally distinct. The lateral region contains thousands of motor proteins but few voltage-gated channels. The basal region, conversely, contains a high number of voltage-gated channels but is devoid of motor proteins. It has been suggested that the cortical cytoskeleton is responsible for maintaining this regional distinction. Towards elucidating the structure of the outer hair cell's electromotile mechanism, we investigated the physical organization of the lateral plasma membrane in living guinea pig outer hair cells by analyzing the distribution pattern of membrane-soluble fluorescent markers within this area, before and after electrical stimulation and with an intact and a disrupted cytoskeleton (Zhang & Kalinec, 2000; Zhang & Kalinec, 2002). Our results suggested that the lateral plasma membrane of guinea pig outer hair cells contain structural microdomains, and that the cytoskeleton does not appear to be playing a major role in maintaining the lateral separation of these distinct molecular regions (Zhang & Kalinec, 2000; Zhang & Kalinec, 2002).
References associated with this Project
Kalinec F, M Zhang, RA Urrutia and G Kalinec, (2000) “Rho GTPases Mediate the Regulation of Cochlear Outer Hair Cell Motility by Acetylcholine”. J.Biol.Chem. 275:28000-28005.
Matsumoto, N. and F. Kalinec. (2005). “Prestin-dependent and prestin-independent motility of guinea pig outer hair cells”. Hear Res. In press. (available online DOI: 10.1016/j.heres.2005.03.030)
Matsumoto, N and F. Kalinec, (2005b). “Extraction of Prestin-Dependent and Prestin-Independent Components from Complex Motile Responses in Guinea Pig Outer Hair Cells”. Biophysical Journal. In press.
Matsumoto, N, S. Chen, and F Kalinec. “Control of the Motile Response of Cochlear Outer Hair Cells by the RhoA/ROCK/LIMK/Cofilin Signaling Cascade”. To be submitted
Zhang M and Kalinec F (2000). “Outer hair cell force-generator mechanism drives lateral displacement of membrane-soluble fluorescent probes. In Recent Developments in Auditory Mechanics. Edited by H. Wada, T Takasaka, K Ikeda, K Oyhama & T Koike. World Scientific, Singapore. Pp. 330-336.
Zhang M and Kalinec F (2002). "Structural microdomains in the basolateral plasma membrane of cochlear outer hair cells". JARO 3:289-301.
Zhang M, G Kalinec, DD Billadeau, R Urrutia and F Kalinec (2002). “ROCK ‘n’ Rho in Outer Hair Cell Motility” In Biophysics of the Cochlea: From Molecules to Models. Edited by Anthony Gummer. World Scientific, Singapore. Pp. 127-135.
Zhang M, G Kalinec, R Urrutia, DD Billadeau, and F Kalinec (2003). “ROCK-dependent and ROCK-independent Control of Cochlear Outer Hair Cell Electromotility”. J.Biol.Chem. 278:35644-35650.
II. Drug-induced hearing loss
Introduction
At the Section on Cell Structure and Function we are exploring the function of the inner ear in order to prevent life-saving drugs and noise from having a noxious effect on the inner ear and killing the outer hair cells. Our goal is to develop of intervention strategies to prevent damage to the outer hair cells.
Some drugs that are critical to maintaining a patient’s health are also toxic to the ear (ototoxic) and can cause hearing loss. In the case of a pregnant woman, the drugs may cross the placental barrier and affect the ear of the developing baby. Since most of the damage from these drugs occurs in the outer hair cells, we are working with different approaches with the goal to identify safe and simple intervention strategies that could prevent sensorineural hearing loss that results form ototoxic agents used in humans.
a) Inner ear cell lines as an in vitro system for ototoxicity screening
Little is known about the cell and molecular basis of drug ototoxicity. Experimental evidence suggests that some well-known ototoxic drugs such as aminoglycoside antibiotics and antineoplastic agents (e.g. cisplatin), cause hearing loss by inducing apoptosis (programmed cell death) of sensory hair cells. Understanding of the cellular and molecular mechanisms underlying drug ototoxicity, however, has been hampered by the limited availability of inner ear tissues and drug side effects on laboratory animals. For example, the effects of systemically administered aminoglycosides take several days or even weeks to develop and there is little control over the amount of drug reaching the affected cells. In order to overcome these limitations, some ototoxicity studies have been performed on explants of cochlear and vestibular organs. Organotypic cultures are not easy to establish, however, and the requirement of several explants for each experimental condition limits the scope of this technique. We envisioned that immortalized cell lines derived from the organ of Corti growing in environments that could be systematically manipulated would be a valuable alternative. If sensitive to ototoxic drugs, such cell lines could facilitate the elucidation of the molecular mechanisms of drug ototoxicity as well as help devise better strategies to prevent ototoxic drug-induced sensorineural hearing loss. Furthermore, in a matter of hours or days, depending on the selected biological assay, many different drugs could be tested simultaneously and at different concentrations, increasing productivity while diminishing costs and avoiding ethical concerns associated with animal research. The cell lines can also be used in automated, high throughput platforms to simultaneously evaluate large numbers of drugs, to determine toxic profiles, and to investigate potential synergistic interactions.
With this idea, we developed several cell lines from mouse cochleae and found that particularly one of them —termed House Ear Institute-Organ of Corti 1 (HEI-OC1)— was extremely sensitive to ototoxic drugs while resistant to non-ototoxic ones. We proposed this cell line as an in vitro system for drug ototoxicity screening (Kalinec et al, 2003) and used it for investigating the cellular and molecular mechanisms activated by ototoxic drugs. The value of HEI-OC1 cells as a tool for investigating drug ototoxicity was immediately recognized, and many laboratories around the world requested cells for performing their own research.
b) Pivotal role of Harakiri (Hrk) in aminoglycoside-induced apoptosis
Experimental evidence accumulated during the past ten years strongly suggests that drug-induced hearing loss is directly associated with apoptosis of OHCs mediated by oxidative stress. Unfortunately, the signaling pathways and molecular effectors mediating in this process remain poorly understood. This gap in the existent knowledge limits the research for new otoprotective agents rather to an empirical matter of trial and error. Mechanistic studies that, by revealing specific molecular pathways involved in ototoxicity provide the conceptual basis for the rational design of therapeutic approaches to prevent this phenomenon, have been hampered by the lack of adequate cellular models. We have used HEI-OC1 cells (Kalinec et al, 2003) for identification and characterization of the signaling pathways activated by aminoglycoside antibiotics and the antineoplastic agent cisplatin. Recent results provided the first evidence indicating that Harakiri, a pro-apoptotic member of the Bcl2 family, would be an important —if not the most important— component of the apoptotic signaling cascade activated by aminoglycosides (Kalinec et al, 2005). Studies with the ototoxic aminoglycoside antibiotics gentamicin, kanamycin, tobramycin, neomycin and streptomycin indicate that all of them increase the expression of Harakiri in HEI-OC1 cells and hair cells of mice and guinea pigs. Moreover, the apoptosis of HEI-OC1 cells induced by these drugs is abolished by preventing Harakiri upregulation. Therefore, our work has identified Harakiri as a crucial molecular target for pharmacological interventions, and set the background for the design of clinical strategies for preventing aminoglycoside ototoxicity.
c) Prevention of drug-induced perinatal hearing loss with L-carnitine
It has long been recognized that ototoxic drugs should be avoided during pregnancy. However, this is not always possible, and the benefits of certain drugs in combating life-threatening diseases may often outweigh the risks. Although ototoxic medication is one of the 10 major risk factors for hearing loss, the incidence of hearing loss in neonates following treatment of the mother with aminoglycoside antibiotics is not well documented. Similarly, there are no published reports of clinical strategies aimed at preventing drug-induced hearing loss in newborns. This incongruity is the central focus of our research efforts.
The primary site of action of most ototoxic agents is the mitochondria of auditory hair cells. Pregnant women readily develop a deficiency in L-carnitine (LCAR), a natural micronutrient and antioxidant required for normal mitochondrial function. In addition, a combination of decreased synthetic capacity, deficient stores and increased losses by the immature renal tubule renders the fetuses and neonates strictly dependent on their mothers to maintain normal LCAR levels. Based on these premises, we have used pregnant guinea pigs as an animal model to investigate the effect of exposure of mothers and newborns to the ototoxic drugs gentamicin, kanamycin and cisplatin during the prenatal period.
These studies are complementary to those performed on HEI-OC1 cells (see above). In vitro data must be corroborated in animal models before moving to clinical trials in human patients. Results in HEI-OC1 cells suggesting that the pro-apoptotic agent Harakiri (Hrk) and the MAP-Kinases ERK (see above) are crucial components of the signaling pathways activated by ototoxic drugs have been further supported by the finding that exposure to gentamicin both activates ERK and increases the expression of Hrk in the cochlear outer hair cells of mothers and newborn guinea pigs.
The results of our studies also suggest that LCAR supplementation might prevent gentamicin- and cisplatin-induced hearing loss and cochlear damage in newborn guinea pigs. Since drug potency, fetal age, and dosage determine the effect of the drug on the fetus, we are now exploring the effect of different doses of these drugs when systemically administered to guinea pigs at different stages of pregnancy. These studies will provide important information to identify the period of cochlear development most sensitive to drug exposure, and to evaluate a simple intervention strategy for preventing drug-induced hearing loss in newborns.
References associated with this Project
Devarajan P, M Savoca, M-S Park, MP Castaneda, N Esteban, G Kalinec, and F Kalinec. “Cisplatin-Induced Apoptosis of Auditory Cells: Role of death receptor and mitochondrial pathways”. Hear.Res. 174:45-54, 2002.
Kalinec GM, P Webster, DJ. Lim and F Kalinec. “A Cochlear Cell Line as an In Vitro System for Drug Ototoxicity Screening”. Audiol.Neurootol. 8(4):177-189, 2003.
So H-S, C Park, H-J Kim, J-H Lee, S-Y Park, J-H Lee, Z-W Lee, H-M Kim, F Kalinec, DJ Lim and R Park. “Protective effect of T-Type calcium Channel blocker flunarizine on cisplatin-induced death of auditory cells”. Hear. Res. 204:127-139, 2005.
Kalinec, G, Fernandez-Zapico, M, Urrutia, R, Esteban-Cruciani, N, Chen, S, and Kalinec, F.: “Pivotal role of harakiri in the induction and prevention of gentamicin-induced hearing loss”. Proc.Natl.Acad.Sci. (USA). In press.
