The remarkable dynamic range of the mammalian inner ear relies on the exceptional sensitivity of hair cells, which are critically dependent on the existence a unique, organ-specific, positive EP (Davis 1957
; Hudspeth 1997
). Processes that are associated with disruption of K+
transport and recycling within the inner ear—and therefore, with the generation of the EP—are associated with hearing loss (Carlisle et al. 1990
; Salt and DeMott 1999
). Recent work studying the knockout of specific K+
channel KCNJ10 (Kir4.1) demonstrates abolition of EP with profound hearing loss, whereas pharmacologic inhibition of KCNJ10 confers reduction in EP and concomitant reduction in hearing thresholds (Marcus et al. 2002
; Nie et al. 2005
; Wangemann et al. 2004
). By far, the most clinically relevant process for neurosensory hearing loss in humans is age-related hearing loss. Age-related hearing loss, or presbycusis, affects one in three persons over 60 years of age and one in two persons over 85 years of age (Gratton and Vazquez 2003
Histopathological evidence exists, from cadaveric evaluation of temporal bones of human patients with presbycusis, that age-related hearing loss can be described by four types of cochlear histopathology: (1) sensory presbycusis involving hair cell loss, (2) neural presbycusis involving loss of spiral ganglion cells and axons, (3) mechanical or conductive presbycusis thought to be conferred by stiffening of the basilar membrane, and (4) metabolic or strial presbycusis, whereby atrophy of the StV is thought to confer hearing loss (Schuknecht and Gacek 1993
). Clinically, however, presbycusis is rarely found to present as one specific audiometric pattern correlating to one specific histopathologic process, but it is commonly considered to be an amalgamation of patterns—and hence, a combination of several processes (Shih 1994
Considerable experimental evidence exists to suggest that age-related hearing loss may be associated with progressive metabolic and structural changes within the lateral wall of the cochlea. Animal models of age-related hearing loss demonstrate progressive alteration of biochemical and structural integrity of the cochlear lateral wall, including reduction in expression and activity of Na,K–ATPase, reduction in expression of NKCC1, involution and atrophy of the stria vascularis, degeneration of spiral ligament fibrocytes, segmental degeneration of capillary vasculature, and decline in EP associated with such changes (Gratton et al. 1996
Gratton and Schulte 1995
; Hequembourg and Liberman 2001
; McGuirt and Schulte 1994
; Sakaguchi et al. 1998
; Schulte and Adams 1989
; Schulte and Schmiedt 1992
; Schulte and Steel 1994
; Spicer et al. 1997
In this series of genetic experiments, these same key ion transport mechanisms were identified and targeted for gene deletion and systematically nulled, individually and in combination, to investigate effects on hearing, cochlear function and structure, and EP generation.
Homozygote deletion of NKCC1 has previously been studied and showed 28% incidence of death at the time of weaning. Those mice that survived demonstrated immediate profound hearing loss without measurable thresholds on ABR (Flagella et al. 1999
). Homozygote deletion of α1
–Na,K–ATPase and α2
–Na,K–ATPase both were uniformly fatal at birth, indicating the critical role that the Na,K pump plays for the viability of mammalian organisms (Moseley et al. 2003
; unpublished data).
Heterozygote mutations of these three critical transporters, on the other hand, are not partially or uniformly fatal, and they display audiometric characteristics analogous to that of mammalian forms of age-related hearing loss. Heterozygote mutation of NKCC1 individually demonstrates a mild to moderate hearing loss across all frequencies early in age that progresses with age. Heterozygote mutation of α1
–Na,K–ATPase and α2
–Na,K–ATPase individually show similar age-progressive hearing loss, comparable in severity to the NKCC1+/−
heterozygote at later age but with an earlier onset of severity. In addition, terminal EP amplitude measurements demonstrate significant depression, but still, existence of a positive EP amplitude in each of the single heterozygote models. This finding is consistent with EP measurements obtained from quiet-aged mammals (Gratton et al. 1996
). The normal appearance of the cochlear duct and organ of Corti from light and scanning electron microscopy imply that changes to hearing function as a result of these genetic manipulations are dynamic or metabolic in origin rather than static or structural.
Considered individually, each of these single heterozygote mutations can serve as a potential genetic and molecular biologic model for age-related hearing loss based on ABR threshold and EP data. Each model is not necessarily equivalent, however, as the earlier disruption in hearing thresholds in the Na,K–ATPase heterozygotes implies a relative primacy of Na,K–ATPase over NKCC1 in the preservation of hearing.
Intermodulation distortion from low-level stimuli is observed only in the NKCC1+/−
single heterozygote model and not measurable in either of the Na,K–ATPase heterozygote models. This divergence between NKCC1 and Na,K–ATPase is not fully understood at this time. One potential mechanism for the effects of heterozygote mutation on DPOAE would involve depression in OHC function and subsequent reduction in DPOAE level secondary to depressed EP amplitude. However, as terminal EP amplitudes are comparable between the three heterozygote groups, there is no reason why the degree of intermodulation distortion produced would be favored for one group over the others. Conversely, as low-level intermodulation distortion is generated by reverse transduction at the OHC, it is plausible that neuronal pathways, rather than strial/lateral wall metabolism, are the dominant site of disruption causing this phenomena. Na,K–ATPase has been identified in high levels both in spiral ganglion neurons and in unmyelinated nerve fibers within the organ of Corti, whereas NKCC1 has been identified only weakly in satellite cells within the spiral ganglion (Crouch et al. 1997
; Schulte and Adams 1989
). This differential expression of Na,K–ATPase over NKCC1 within the cochlear neuronal pathways makes the latter proposition more plausible. It is evident from these data, however, that generation of low-level stimuli intermodulation distortion is more sensitive to disruption in Na,K–ATPase activity than NKCC1 activity.
Functional results from double heterozygote mutations of NKCC1 with either α1
–Na,K–ATPase and α2
–Na,K–ATPase demonstrate even more fascinating results. The
double heterozygote mice exhibit age-related ABR threshold shifts and hearing loss comparable to each of the single heterozygote conditions NKCC1+/−
, and α2
, but with a delayed progression of severity compared to the single heterozygotes.
double heterozygote mice demonstrate an even more remarkable effect of ABR threshold stability and complete hearing preservation with age. The
double heterozygote mice proceeded to a profound hearing loss after 1 year of age, whereas the
double heterozygote mice maintained ABR thresholds equivalent to age-matched controls at all times. Mean terminal EP amplitude of the
group was preserved, suggesting preservation of the mechanisms for EP generation within the cochlear lateral wall.
Intermodulation distortion was present in both double heterozygote models, only at low frequencies for
but throughout the frequency range for
. This is contrasted with the complete absence of low-level stimuli DPOAE in α1
Modulation of the activity of NKCC1 simultaneously with Na,K–ATPase appears to confer a beneficial crosstalk or rescue effect within the cochlear metabolic architecture over that of either ion transporter alone: Reverse transduction and OHC function are reinstated, and age-related hearing loss is delayed. In addition, the specific combination of NKCC1 modulation with α2–Na,K–ATPase appears to confer the greatest advantage, as EP is preserved and age-related hearing loss is completely mitigated.
The results of these double heterozygote experiments have implications beyond modeling for age-related hearing loss, as they help to verify predictions of the roles of NKCC1, α1–Na,K–ATPase, and α2–Na,K–ATPase in K+ cycling within the inner ear. Based on the two cell model of EP generation, these transport proteins serve in balancing the K+ flux through the lateral wall, α2–Na,K–ATPase + NKCC1 in spiral ligament absorption from the perilymph at types II, IV, and V fibrocytes, and α1–Na,K–ATPase + NKCC1 in strial secretion back into the endolymph through the MC (Fig. ). Downregulation of one mechanism causes disruption in EP generation and hearing loss, but coincident downregulation of both mechanisms—as is done in the double heterozygote models—results in stability of hearing, presumably from downregulated but balanced K+ flux and, thus, successful regulation of EP generation over time. Based on the single heterozygote experiments, Na,K–ATPase appears to be the more relevant rate-limiting factor over NKCC1, and based on the double heterozygote experiments, α2–Na,K–ATPase appears to be the more relevant rate-limiting factor over α1–Na,K–ATPase.
FIG. 8 K+ cycling and ion transport in the inner ear—diagrammatic representation of the two-cell model for the generation of EP in the StV. We have targeted the NKCC1, α1–Na,K–ATPase, and α2–Na,K–ATPase (more ...)
The development of this multiple targeted gene deletion framework continues to expand upon our knowledge and understanding of the complex biochemical machinery necessary for the development and maintenance of the proper intracochlear environment requisite for hearing and balance. It is clear that each of these key ion transporters is critical to the proper functioning of this machinery. The relative primacy of Na,K–ATPase over NKCC1 within this milieu, and the implication of spiral ligament α2–Na,K–ATPase as the rate-limiting element for K+ flux within the lateral wall, is proposed based on the functional results of the series of single and compound knockout experiments performed.
From these results, we gain a better understanding of the pathophysiology of strial-related mechanisms of hearing loss. Further, we identify multiple potential loci for intervention in certain forms of hearing loss imparted by ion transport defects, such as ototoxicity and age-related hearing loss. For example, it may be conceivable to counter the effects of a diuretic-induced ototoxicity at NKCC1 by administering a titrated dose of a cardiac glycoside to differentially inhibit Na,K–ATPase. As genetic identification of an individual’s hearing loss becomes reality, it may be possible to pharmacologically or genetically counterbalance an inherited predisposition to age-related hearing loss. Should a patient have an inherited defect in one of these ion transporters within the ear, it is conceivable that future therapy may involve genetic means of upregulating the gene for that transporter, or, perhaps more elegantly, downregulating the gene for its counterbalancing transporter. In a more general sense then, if inhibition or downregulation at one of these K+ transporters represents a molecular site of hearing loss, then deliberate inhibition at the counterbalancing site may provide a mechanism to preserve hearing or mitigating the hearing loss that would otherwise ensue.