Cardiac Na/Ca Exchange Suppression: A Late-Breaking Knockout Story Showing That There Is No Free Lunch.

At each heartbeat, Ca2+ enters the cardiac myocyte via L‐type Ca current (ICa), triggering additional Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors. In the steady state, the SR Ca pump must resequester the exact amount of Ca2+ that was released and the Na/Ca exchange (NCX) is almost entirely responsible for extruding all of the Ca2+ that entered, mainly by ICa. , Although NCX is critical for this Ca2+ flux balance and preventing acute cellular Ca2+ overload, digitalis‐induced inotropy takes advantage of this relationship to raise myocyte Ca2+ by inhibiting the Na+/K+‐ATPase to increase intracellular [Na+], which limits Ca2+ extrusion via NCX. The downside of this Ca2+ loading is that it can cause arrhythmogenic diastolic Ca2+ leak and NCX itself carries the transient inward current that causes delayed afterdepolarizations (DADs), which can trigger ectopic action potentials (APs) and tachyarrhythmias, especially in heart failure.

The critical role of NCX for cardiac myocyte Ca2+ extrusion made it a complete surprise that the initial cardiac‐specific NCX1 knockout mouse was viable into adulthood. Although these NCX1‐knockout (KO) mice did not fare well long‐term, follow‐up studies revealed remarkable developmental adaptations to limit Ca2+ influx via Ca2+ current, without apparent upregulation of the plasma membrane Ca2+‐ATPase (the only other known Ca2+ extrusion mechanism)¨. Three key factors limited Ca2+ influx in these NCX‐KO myocytes: (1) reduced AP duration (already short in mouse) mediated by (2) an increased transient outward K+ current (Ito), thereby abbreviating ICa duration, and (3) decreased ICa density that appeared to be attributable to local elevation of [Ca2+]i in the junctional cleft and Ca2+‐dependent inactivation of Ca2+ channels.

Inducible NCX1 Knockdown, Homeostatic Compensations, and Protective Effects

In this issue of the Journal of the American Heart Association (JAHA), Lotteau et al circumvent the apparent developmental adaptations by acute knockdown of NCX with a tamoxifen‐sensitive Mer‐Cre‐Mer promoter, allowing inducible knockdown by breeding with NCX1 exon 11 floxed mice. Intraperitoneal administration of hydrotamoxifen (40 mg/kg) resulted in NCX protein expression knockdown by 95% in 1 week and 98% in 4 weeks, allowing assessment of early and later adaptations in adult hearts. At 1 week, with 95% of NCX gone, there was no change in heart weight, ejection fraction, fibrosis, AP duration, or Ito, but already a reduction in ICa and increases in myocyte diastolic and systolic [Ca2+]i, Ca2+ waves, and Ca+‐calmodulin dependent protein kinase II (CaMKII) activation, as well as an increase in plasma membrane Ca2+‐ATPase expression. However, the increased Ca2+ waves did not increase DADs or spontaneous APs, consistent with the role of NCX in mediating DADs and triggered APs and thus their suppression in the NCX KO.

During the next 3 weeks, these NCX‐KO mice develop cardiac and myocyte hypertrophy, interstitial fibrosis, increased Ito, and a virtual abolition of DADs in parallel with the disappearance of the observable caffeine‐induced myocyte NCX current. In addition, both 1 and 4 weeks after NCX knockout, the hearts were partially protected from the damage caused by reperfusion after a 20‐minute period of global ischemia, with infarct size reduced by ≈50% in the 4‐week group. That also makes mechanistic sense, because the increase in [Na+]i during ischemia and rapid recovery of intracellular pH during reperfusion are known to cause dramatic acute myocyte Ca2+ overload mediated by [Na+]i‐dependent Ca2+ influx via NCX.

This all sounds really great! But even without any ischemic challenge, the NCX‐KO mice start dying rapidly 5 weeks after tamoxifen, and 75% die in the next 5 weeks. So what is going on? Reducing NCX prevents some major pathophysiological problems, arrhythmogenic DADs, and ischemia‐reperfusion injury, so why do the mice end up dying?

The Dark Side of NCX Knockdown

The results in this inducible NCX model are interesting in revealing how effectively the heart compensates for the loss of a major physiological player, but how in the end there is still a major price to pay. Reduced NCX function prevents cellular Ca2+ extrusion, tending to cause cellular Ca2+ overload. The latter can easily cause cell death, so the body rapidly brings to bear major and effective compensations. In the study by Lotteau et al, the compensations included reduced ICa attributable to Ca2+‐dependent Ca2+ channel inactivation and reduced AP duration, likely attributable to K+‐current upregulation (both of which decrease Ca2+ entry) on one hand; and increased removal of cytosolic Ca2+ via enhanced SR uptake through upregulation of the SR Ca2+‐ATPase and enhanced transport to the extracellular space across the sarcolemma via the plasma membrane Ca2+‐ATPase pump. However, these compensations cannot fully prevent the consequences of cellular Ca2+ loading, which they only partly offset. One week after tamoxifen administration, both diastolic and systolic cytosolic [Ca2+] are elevated. Three weeks later, at 4 weeks after tamoxifen, cytosolic [Ca2+] is no longer elevated, but SR Ca2+ content is now increased: presumably, more effective SR Ca2+ uptake removes excess Ca2+ from the cytosol, but at the cost of increased SR Ca2+ loading.

Nuclear Ca2+ content is a key regulator of cardiac gene transcription and driver of adverse remodeling. , A key regulator of nuclear Ca2+ content is the nuclear envelope, a double‐bilayer structure that is in direct continuity with the SR. It is therefore highly likely that the compensatory SR Ca2+‐ATPase upregulation and SR Ca2+ loading that cannot be extruded without NCX function result in nuclear Ca2+ loading and the activation of remodeling programs. This idea is consistent with the RNA‐sequencing analysis in the study by Lotteau et al, which showed that only 182 transcripts had changed at 1 week, but 2699 had changed by 4 weeks. It would be of great interest to measure nuclear [Ca2+] as a function of time in inducible NCX KO mice and to relate the changes to alterations in gene expression, cardiac remodeling indexes, and more precise observations on the causes of death in these animals.

Clinical Relevance

These observations might have direct clinical relevance. Small molecules that block NCX1 have been under development for many years, based on evidence that they suppress DAD‐related arrhythmias and ischemia‐reperfusion injury. However, by inhibiting NCX1, they might engage compensatory mechanisms that ultimately promote adverse remodeling and enhance longer‐term mortality, as occurs in NCX1 KO mice.

It is possible that there may be levels of NCX1 inhibition that do not produce such consequences. Relatively normal myocyte Ca2+ handling might be maintained by the compensations noted in the study by Lotteau et al, with a 30% to 50% reduction in NCX current amplitude, whereas this degree of DAD suppression may virtually abolish DAD‐triggered APs. That is because the relationship between [Ca2+]i and DAD amplitude is highly nonlinear, and a certain threshold amplitude is required for AP induction. Although the poor long‐term prognosis of the complete NCX‐KO is notable, it would be interesting to know if a heterozygous NCX1‐KO mouse would be protected from long‐term pathological changes. In addition, the reliability with which greater levels of inhibition could be avoided in patients is a critical question, given the substantial variability in drug pharmacokinetics and pharmacodynamics that is typical of clinical populations.

Another long‐standing question that began with the surprising initial NCX‐KO mouse result is, would larger mammals equally tolerate NCX1‐KO? That is, the mouse (versus rabbit or human) has an extremely short AP, with less Ca2+ influx via ICa, and therefore much less Ca2+ that must be extruded by NCX. Would the same adaptations suffice in humans, in whom transsarcolemmal Ca2+ fluxes are more important? Moreover, the observed upregulation of NCX1 in rabbit and human heart failure may be adaptive (when SR Ca2+ content and release may be impaired). , That is, in human and rabbit HF with reduced SR Ca2+ release and elevated [Na+]i, NCX actually mediates some Ca2+ entry during the AP to support contraction. , , In addition, removal of all the Ca2+ that entered via ICa plus NCX may require greater NCX function to remove the entering Ca2+, because in heart failure the high [Na+]i slows Ca2+ extrusion by NCX and the longer AP duration shortens the diastolic time for Ca2+ extrusion. , , Would reducing NCX function in heart failure further impair diastolic cardiac function?

Conclusions

The study by Lotteau et al is an important contribution to the literature, which shows that the powerful adaptations previously noted in models involving NCX KO from birth are also noted when NCX expression is strongly suppressed at adulthood in an inducible KO model, albeit with some differences in the details of compensatory responses. Furthermore, this work shows that although the associated adaptations prevent major Ca2+ overload, the animals nevertheless eventually get sick and die. These observations urge caution in the development of therapeutic approaches that target pathological consequences related to NCX activity: with cellular Ca2+ handling, as in many areas of life, there is no such thing as a free lunch and potentially disastrous adverse consequences of what seems like a good idea have to be seriously considered.

Disclosures

None.