Randomized Controlled Trial of the Hemodynamic Effects of Empagliflozin in Patients With Type 2 Diabetes at High Cardiovascular Risk: The SIMPLE Trial.

Treatment with the sodium-glucose cotransporter 2 inhibitor (SGLT-2i) empagliflozin significantly reduces cardiovascular events in patients with type 2 diabetes (T2D); however, the mechanisms behind the reduction in cardiovascular (CV) events are unknown. We investigated whether SGLT-2i treatment affected central hemodynamics during rest and exercise in 34 patients with diabetes in this investigator-initiated, randomized, placebo-controlled, double-blinded trial. The primary end point was change in pulmonary capillary wedge pressure (PCWP) at a submaximal ergometer workload (25 W) after 13 weeks of SGLT-2i treatment (25 mg once daily) compared with placebo. Secondary end points included changes in resting hemodynamics. Baseline and follow-up hemodynamic assessments were performed at rest, submaximal exercise (25 W), and peak exercise using right heart catheterization. Treatment with empagliflozin for 13 weeks in patients with T2D at high CV risk did not reduce left heart filling pressure more than placebo at submaximal exercise. At rest, we observed that empagliflozin reduced PCWP at a magnitude of clinical significance.

RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells.

Cav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary ß3 subunit, RIM2α alone (Cav1.342A) or RIM2α/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored ß2 variants (ß2a, ß2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel's activation threshold toward more hyperpolarized potentials. Taken together, the association with "slow" ß subunits (ß2a, ß2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function.

Design of a randomised controlled trial of the effects of empagliflozin on myocardial perfusion, function and metabolism in type 2 diabetes patients at high cardiovascular risk (the SIMPLE trial).

INTRODUCTION: A diagnosis of type 2 diabetes (T2D) more than doubles the risk of cardiovascular disease (CVD), with heart failure (HF) being one of the most common complications with a severe prognosis. The landmark Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Paitients (EMPA-REG OUTCOME) study demonstrated that treatment with the sodium glucose cotransporter-2 (SGLT-2) inhibitor empagliflozin rapidly and significantly reduces CVD mortality and admission rates for HF. However, the mechanisms behind this reduction in clinical events are unknown.This study was designed to investigate the effects of the SGLT-2 inhibitor empagliflozin on myocardial perfusion and function in patients with T2D and high CVD risk. METHODS AND ANALYSIS: In this investigator-initiated, randomised, double-blind controlled clinical trial, 92 patients with T2D and established CVD or high CVD risk will be randomised to treatment with empagliflozin 25 mg or a matching placebo for 13 weeks. The primary outcome measure is change in myocardial flow reserve measured quantitatively by Rubidium-82 position emission tomography. In a substudy, invasive haemodynamics at rest and during exercise will be measured at baseline and following the intervention, using right heart catheterisation. ETHICS AND DISSEMINATION: The study protocol (v7, 02/08/2018) has been approved by the Ethics Committee of the Capital Region, Danish Data Protection Board and the Danish Medicines Agency, and it will be monitored according to the Good Clinical Practice regulations from the International Conference on Harmonization. The results be submitted to international peer-reviewed journals and be presented at conferences. The data will be made available to the public via EudraCT and www.clinicaltrials.gov. TRIAL REGISTRATION NUMBER: NCT03151343.

Effect of the mineralocorticoid receptor antagonist eplerenone on liver fat and metabolism in patients with type 2 diabetes: A randomized, double-blind, placebo-controlled trial (MIRAD trial).

AIM: To investigate whether the mineralocorticoid receptor antagonist eplerenone has beneficial effects on liver fat and metabolism in patients with type 2 diabetes (T2D), the mineralocorticoid receptor antagonist in type 2 diabetes (MIRAD) trial. MATERIAL AND METHODS: In this 26-week, double-blind, randomized, placebo-controlled trial, we enrolled 140 patients with T2D and high risk of cardiovascular disease. Patients were randomized 1:1 to either eplerenone with a target dose of 200 mg/day for patients with estimated glomerular filtration rate (eGFR) of 60 mL/min per 1.73 m2 or more and 100 mg/day for patients with eGFR between 41 and 59 mL/min per 1.73 m2 or placebo. The primary outcome measure was change in liver fat by proton magnetic resonance spectroscopy at week 26 from baseline; secondary outcomes were changes in metabolism, and safety by incident hyperkalaemia. RESULTS: No changes in liver fat in the eplerenone group 0.91% (95% CI -0.57 to 2.39) or the placebo group -1.01% (-2.23 to 0.21) were found. The estimated absolute treatment difference was 1.92% (-3.81 to 0.01; P = 0.049). There was no beneficial impact on supporting secondary outcome variables of metabolism as fat mass distribution, lipid metabolism or insulin resistance. Despite a high dosage of eplerenone 164 versus 175 mg in patients treated with placebo (P = 0.228), the number of patients with incident hyperkalaemia (≥5.5 mmol/L) was low, with six in the eplerenone versus two in the placebo group (P = 0.276). CONCLUSION: The addition of high doses of eplerenone to background antidiabetic and antihypertensive therapy does not show beneficial effects on liver fat and metabolism in patients with T2D.

Single lead atrial vs. dual chamber pacing in sick sinus syndrome: extended register-based follow-up in the DANPACE trial.

AIMS: The DANPACE trial randomized patients with sick sinus syndrome (SSS) to single lead atrial (AAIR) or dual chamber (DDDR) pacemaker (PM). After 5 years follow-up, no difference in overall survival, stroke or heart failure (HF) was observed, whereas risk of atrial fibrillation (AF) and PM reoperation were increased in the AAIR group. The present study aimed to investigate very long term risk of death, AF hospitalization, stroke, HF and rate of change in pacing mode using national register-based data. METHODS AND RESULTS: The study population consisted of all 1384 patients included at Danish PM centres in the DANPACE trial randomized to AAIR (n = 696) or DDDR (n = 688). Long-term follow-up data was obtained from Danish national registers. Analysis was intention-to-treat. results: During mean follow-up of 8.9 years, 413 patients (59.3%) died in the AAIR-group compared to 367 (53.3%) in the DDDR-group (adjusted hazard ratio 1.03; 95% confidence interval 0.90-1.19; P = 0.65). We observed no difference in risk of AF hospitalization, stroke or HF. During extended follow-up, annual rate of pacing mode change to DDDR in the AAIR group was 4.5%, and higher than the 2.3% observed during trial conduct. CONCLUSION: This register-based long-term follow-up study indicates that there is no difference in mortality among patients with SSS randomized to AAIR or DDDR pacing, even with very long follow-up. Nor is there any difference in risk of AF hospitalization, stroke or HF. The higher rate of pacing mode-change to DDDR in the AAIR group suggests a different management of patients with an AAIR PM after the DANPACE trial.

Autonomous functions of murine thyroid hormone receptor TRα and TRß in cochlear hair cells.

Thyroid hormone acts on gene transcription by binding to its nuclear receptors TRα1 and TRß. Whereas global deletion of TRß causes deafness, global TRα-deficient mice have normal hearing thresholds. Since the individual roles of the two receptors in cochlear hair cells are still unclear, we generated mice with a hair cell-specific mutation of TRα1 or deletion of TRß using the Cre-loxP system. Hair cell-specific TRß mutant mice showed normal hearing thresholds but delayed BK channel expression in inner hair cells, slightly stronger outer hair cell function, and slightly reduced amplitudes of auditory brainstem responses. In contrast, hair cell-specific TRα mutant mice showed normal timing of BK channel expression, slightly reduced outer hair cell function, and slightly enhanced amplitudes of auditory brainstem responses. Our data demonstrate that TRß-related deafness originates outside of hair cells and that TRα and TRß play opposing, non-redundant roles in hair cells. A role for thyroid hormone receptors in controlling key regulators that shape signal transduction during development is discussed. Thyroid hormone may act through different thyroid hormone receptor activities to permanently alter the sensitivity of auditory neurotransmission.

Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells.

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.

Biochemical screening of 504,049 newborns in Denmark, the Faroe Islands and Greenland--experience and development of a routine program for expanded newborn screening.

Expanded newborn screening for selected inborn errors of metabolism (IEM) in Denmark, the Faroe Islands and Greenland was introduced in 2002. We now present clinical, biochemical, and statistical results of expanded screening (excluding PKU) of 504,049 newborns during nine years as well as diagnoses and clinical findings in 82,930 unscreened newborns born in the same period. The frequencies of diagnoses made within the panel of disorders screened for are compared with the frequencies of the disorders in the decade preceding expanded newborn screening. The expanded screening was performed as a pilot study during the first seven years, and the experience obtained during these years was used in the development of the routine neonatal screening program introduced in 2009. Methods for screening included tandem mass spectrometry and an assay for determination of biotinidase activity. A total of 310 samples from 504,049 newborns gave positive screening results. Of the 310 results, 114 were true positive, including results from 12 newborns in which the disease in question was subsequently diagnosed in their mothers. Thus, the overall frequency of an IEM in the screening panel was 1:4942 (mothers excluded) or 1:4421 (mothers included). The false positive rate was 0.038% and positive predictive value 37%. Overall specificity was 99.99%. All patients with true positive results were followed in The Center for Inherited Metabolic Disorders in Copenhagen, and the mean follow-up period was 45 months (range 2109 months). There were no deaths among the 102 children, and 94% had no clinically significant sequelae at last follow-up. Our study confirms the higher frequency of selected IEM after implementation of expanded newborn screening and suggests an improved outcome for several disorders. We argue that newborn screening for these disorders should be standard of care, though unresolved issues remain, e.g. about newborns with a potential for remaining asymptomatic throughout life. Well organized logistics of the screening program from screening laboratory to centralized, clinical management is important.

Critical role for cochlear hair cell BK channels for coding the temporal structure and dynamic range of auditory information for central auditory processing.

Large conductance, voltage- and Ca(2+)-activated K(+) (BK) channels in inner hair cells (IHCs) of the cochlea are essential for hearing. However, germline deletion of BKα, the pore-forming subunit KCNMA1 of the BK channel, surprisingly did not affect hearing thresholds in the first postnatal weeks, even though altered IHC membrane time constants, decreased IHC receptor potential alternating current/direct current ratio, and impaired spike timing of auditory fibers were reported in these mice. To investigate the role of IHC BK channels for central auditory processing, we generated a conditional mouse model with hair cell-specific deletion of BKα from postnatal day 10 onward. This had an unexpected effect on temporal coding in the central auditory system: neuronal single and multiunit responses in the inferior colliculus showed higher excitability and greater precision of temporal coding that may be linked to the improved discrimination of temporally modulated sounds observed in behavioral training. The higher precision of temporal coding, however, was restricted to slower modulations of sound and reduced stimulus-driven activity. This suggests a diminished dynamic range of stimulus coding that is expected to impair signal detection in noise. Thus, BK channels in IHCs are crucial for central coding of the temporal fine structure of sound and for detection of signals in a noisy environment.

Ergic2, a brain specific interacting partner of Otoferlin.

BACKGROUND: Otoferlin, a postulated calcium sensor of 230 kDa, was proposed to trigger calcium dependent fusion of vesicles with plasma membrane in the ribbon synapses of cochlear IHCs. Otoferlin's interaction with Rab8b and Myo6, proteins involved in the intracellular membrane trafficking, extended the previous hypothesis assigning Otoferlin an additional role in trans-Golgi trafficking. Here, we present another Otoferlin binding partner, Ergic2, a protein with a still unknown function but presenting sequence homology to other proteins involved in ER/Golgi vesicle trafficking. METHODS: Novel binding partners of Otoferlin were searched by yeast two-hybrid screening in a rodent cochlear cDNA library (P3-P15). RT-PCR, western blot, immunohistochemistry staining and co-immunoprecipitation were applied to analyze and confirm an interaction between Ergic2 and Otoferlin. RESULTS: The Y-2-H screening, using baits covering parts of Otoferlin's C2D domain, identified Ergic2 as an interacting protein for Otoferlin. Both are co-expressed (mRNA and protein level) in rodent cochlea and brain before- and after-onset of hearing. By RT-PCR Ergic2 was detected in cochlear IHCs and OHCs and in brain regions where Otoferlin is known to be present. Co-localization studies revealed an overlap of Ergic2 and Otoferlin signals in IHCs and neurons of cerebral cortical layer I making Ergic2 the promising binding candidate. However, while Ergic2 was co-precipitated by an anti-Otoferlin antibody in protein lysates from murine brain, this specific protein interaction was not detected in cochlea. CONCLUSION: Our new data on Otoferlin's interactome suggest that Otoferlin can form different, tissue-specific protein complexes.

MCAD deficiency in Denmark.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common defect of fatty acid oxidation. Many countries have introduced newborn screening for MCADD, because characteristic acylcarnitines can easily be identified in filter paper blood spot samples by tandem mass spectrometry (MS/MS), because MCADD is a frequent disease, and because of the success of early treatment initiated before clinical symptoms have emerged. In Denmark we have screened 519,350 newborns for MCADD by MS/MS and identified 58 affected babies. The diagnosis of MCADD was confirmed in all 58 newborns by mutation analysis. This gives an incidence of MCADD detected by newborn screening in Denmark of 1/8954. In sharp contrast to this we found that the incidence of clinically presenting MCADD in Denmark in the 10 year period preceding introduction of MS/MS-based screening was only 1 in 39,691. This means that four times more newborns with MCADD are detected by screening than what is expected based on the number of children presenting clinically in an unscreened population. The mutation spectrum in the newborns detected by screening is different from that observed in clinically presenting patients with a much lower proportion of newborns being homozygous for the prevalent disease-causing c.985A>G mutation. A significant number of the newborns have genotypes with mutations that have not been observed in patients detected clinically. Some of these mutations, like c.199T>C and c.127G>A, are always associated with a milder biochemical phenotype and may cause a milder form of MCADD with a relatively low risk of disease manifestation, thereby explaining part of the discrepancy between the frequency of clinically manifested MCADD and the frequency of MCADD determined by screening. In addition, our data suggest that some of this discrepancy can be explained by a reduced penetrance of the c.985A>G mutation, with perhaps only 50% of c.985A>G homozygotes presenting with disease manifestations. Interestingly, we also report that the observed number of newborns identified by screening who are homozygous for the c.985A>G mutation is twice that predicted from the estimated carrier frequency. We therefore redetermined the carrier frequency in a new sample of 1946 blood spots using a new assay, but this only confirmed that the c.985A>G carrier frequency in Denmark is approximately 1/105. We conclude that MCADD is much more frequent than expected, has a reduced penetrance and that rapid genotyping using the initial blood spot sample is important for correct diagnosis and counseling.

cGMP-Prkg1 signaling and Pde5 inhibition shelter cochlear hair cells and hearing function.

Noise-induced hearing loss (NIHL) is a global health hazard with considerable pathophysiological and social consequences that has no effective treatment. In the heart, lung and other organs, cyclic guanosine monophosphate (cGMP) facilitates protective processes in response to traumatic events. We therefore analyzed NIHL in mice with a genetic deletion of the gene encoding cGMP-dependent protein kinase type I (Prkg1) and found a greater vulnerability to and markedly less recovery from NIHL in these mice as compared to mice without the deletion. Prkg1 was expressed in the sensory cells and neurons of the inner ear of wild-type mice, and its expression partly overlapped with the expression profile of cGMP-hydrolyzing phosphodiesterase 5 (Pde5). Treatment of rats and wild-type mice with the Pde5 inhibitor vardenafil almost completely prevented NIHL and caused a Prkg1-dependent upregulation of poly (ADP-ribose) in hair cells and the spiral ganglion, suggesting an endogenous protective cGMP-Prkg1 signaling pathway that culminates in the activation of poly (ADP-ribose) polymerase. These data suggest vardenafil or related drugs as possible candidates for the treatment of NIHL.

Developmental regulation of glycine receptors at efferent synapses of the murine cochlea.

Efferent olivocochlear feedback innervation modulates the stream of auditory information from cochlea to brainstem by regulating auditory nerve activity and controlling the contribution of cochlear outer hair cells to basilar membrane motion. In our previous work, we gave a first description of glycine receptors (GlyRs) in the rat cochlea indicating a possible localization at efferent cochlear synapses. Here, we analyze the developmental regulation of GlyR transcripts and protein within the developing murine organ of Corti (postnatal days P0-P21). Using quantitative RT-PCR, GlyRα1 and α2 were identified as the predominant GlyRα subunit transcripts before the onset of hearing (

Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness.

Deafness is genetically very heterogeneous and forms part of several syndromes. So far, delayed rectifier potassium channels have been linked to human deafness associated with prolongation of the QT interval on electrocardiograms and ventricular arrhythmia in Jervell and Lange-Nielsen syndrome. Ca(v)1.3 voltage-gated L-type calcium channels (LTCCs) translate sound-induced depolarization into neurotransmitter release in auditory hair cells and control diastolic depolarization in the mouse sinoatrial node (SAN). Human deafness has not previously been linked to defects in LTCCs. We used positional cloning to identify a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Ca(v)1.3 LTCCs, in two consanguineous families with deafness. All deaf subjects showed pronounced SAN dysfunction at rest. The insertion of a glycine residue in a highly conserved, alternatively spliced region near the channel pore resulted in nonconducting calcium channels that had abnormal voltage-dependent gating. We describe a human channelopathy (termed SANDD syndrome, sinoatrial node dysfunction and deafness) with a cardiac and auditory phenotype that closely resembles that of Cacna1d(-/-) mice.

Modulation of Cav1.3 Ca2+ channel gating by Rab3 interacting molecule.

Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Ca(v)1.3 voltage-gated L-type Ca(2+) channels. Their voltage- and Ca(2+)-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2alpha (RIM2alpha) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Ca(v)1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the beta-subunit of the channel complex and RIM-induced slowing of both Ca(2+)- and voltage-dependent inactivation of Ca(v)1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Ca(v)1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Ca(v)1.3 gating kinetics in immature IHCs.

Deafness in TRbeta mutants is caused by malformation of the tectorial membrane.

Thyroid hormone receptor beta (TRbeta) dysfunction leads to deafness in humans and mice. Deafness in TRbeta(-/-) mutant mice has been attributed to TRbeta-mediated control of voltage- and Ca(2+)-activated K(+) (BK) channel expression in inner hair cells (IHCs). However, normal hearing in young constitutive BKalpha(-/-) mutants contradicts this hypothesis. Here, we show that mice with hair cell-specific deletion of TRbeta after postnatal day 11 (P11) have a delay in BKalpha expression but normal hearing, indicating that the origin of hearing loss in TRbeta(-/-) mutant mice manifested before P11. Analyzing the phenotype of IHCs in constitutive TRbeta(-/-) mice, we found normal Ca(2+) current amplitudes, exocytosis, and shape of compound action potential waveforms. In contrast, reduced distortion product otoacoustic emissions and cochlear microphonics associated with an abnormal structure of the tectorial membrane and enhanced tectorin levels suggest that disturbed mechanical performance is the primary cause of deafness resulting from TRbeta deficiency.

Persistence of Ca(v)1.3 Ca2+ channels in mature outer hair cells supports outer hair cell afferent signaling.

Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca2+ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca2+ channel currents (charge carrier, 10 mM Ba2+) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from approximately 170 to approximately 50 pA at approximately the onset of hearing. Ba2+ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba2+ current amplitude of apical OHCs was 58 +/- 9 pA (n = 20, P19-P30) compared with that of the inner hair cells (IHCs) of 181 +/- 50 pA (n = 24, P17-P30). Properties of Ba2+ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by +9 mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Ca(v)1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Ca(v)1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Ca(v)1.3 and CtBP2/RIBEYE protein strongly suggests a role for Ca(v)1.3 channels in exocytosis of mature OHCs.

Thyroid hormone deficiency affects postnatal spiking activity and expression of Ca2+ and K+ channels in rodent inner hair cells.

Thyroid hormone (TH) is essential for the development of hearing. Lack of TH in a critical developmental period from embryonic day 17 to postnatal day 12 (P12) in rats and mice leads to morphological and functional deficits in the organ of Corti and the auditory pathway. We investigated the effects of TH on inner hair cells (IHCs) using patch-clamp recordings, capacitance measurements, and immunocytochemistry in hypothyroid rats and athyroid Pax8-/- mice. Spontaneous and evoked Ca2+ action potentials (APs) were present in control IHCs from P3-P11 rats and vanished in parallel with the expression of a rapidly activating Ca2+- and voltage-activated K+ (BK) conductance. IHCs of hypothyroid rats and athyroid Pax8-/- mice displayed APs until the end of the third postnatal week because of threefold elevated Ca2+ currents and missing expression of BK currents. After the fourth postnatal week, some IHCs showed BK currents whereas adjacent IHCs did not, demonstrated by electrophysiology and immunocytochemistry. To test whether the prolonged spiking activity during TH deficiency may be transmitted at IHC synapses, capacitance measurements were performed in parallel to analysis of otoferlin expression, a protein thought to play an essential role in exocytosis of IHCs. Strikingly, otoferlin was absent from IHCs of hypothyroid rats but not of Pax8-/- mice, although both cell types showed exocytosis with an efficiency typical for immature IHCs. These results demonstrate for the first time a TH-dependent control of IHC spiking activity before the onset of hearing attributable to effects of TH on Ca2+ and BK channels. Moreover, they question an indispensable role of otoferlin for exocytosis in IHCs.