Ca2+ Signaling Domains Responsible For Cardiac Hypertrophy and Arrhythmias
See related articles, pages 514–521 and 522–530
Ca2+ activates and regulates multiple processes in every cell type. In the mammalian heart, cyclic fluctuations in cytosolic [Ca2+] induce and regulate the strength of cardiac contraction (termed “contractile” [Ca2+]). In addition, changes in Ca2+ appear to be centrally involved in normal and pathological signaling (termed “signaling” [Ca2+]) that regulates myocyte growth, hypertrophy, apoptosis, and necrosis.1 Whether or not contractile and signaling [Ca2+] are derived from common or distinct sources and are constrained to unique cellular microdomains is not established.2 What is clear is that cardiovascular diseases including hypertension and myocardial infarction are associated with alterations in contractile and possibly signaling [Ca2+] that are centrally involved in pathological cardiac hypertrophy, heart failure progression,1 and lethal cardiac arrhythmias.3 Defining the sources of signaling Ca2+ involved in the induction of pathological hypertrophy and the bases of dysregulated contractile [Ca2+] in cardiovascular disease should identify novel ways to treat heart disease.
In this issue of Circulation Research, 2 independent reports address fundamental aspects of alterations in signaling and contractile [Ca2+]. Chiang et al4 have studied the idea that Ca2+ influx through voltage operated α1H (CaV3.2) T-type Ca2+ channels (TTCCs) is the source of the signaling [Ca2+] that activates the calcineurin (Cn)-NFAT (nuclear factor of activated T cells) signaling cascade and induces pathological cardiac hypertrophy in pressure overload. In a separate report, Terentyev et al5 explore the idea that microRNA (miR)-1, a muscle-specific microRNA that increases in abundance in cardiac disease,6 causes dysregulated contractile [Ca2+] and induces single cell arrhythmias. These 2 reports are provocative and, if independently confirmed, will have identified novel mechanisms for abnormalities in the signaling and contractile [Ca2+] that cause hypertrophy and sudden death.
Almost 20 years ago, we7 and others8 showed that TTCCs are reexpressed in adult ventricular myocytes after pressure overload. TTCCs are expressed in fetal/neonatal heart but are not normally found in the adult ventricular myocyte. We speculated that the Ca2+ influx through these channels was involved in the induced cardiac hypertrophy.7 The report by Chiang et al4 explores this idea in TTCC knockout (KO) mouse models. There are 3 TTCC genes, and 2 (α1G [CaV3.1] and α1H [CaV3.2]) are found in the heart.9 CaV3.110 and 3.2 KO11 animals, each of which is viable with modest basal phenotypes,10,11 were used. The authors make the provocative observation that thoracic aortic constriction (TAC) induces cardiac hypertrophy in the CaV3.1 KO and control animals, but not in CaV3.2 KO. CaV3.2 KO animals had similar degrees of pressure overload after TAC, documenting a similar degree of stress. The inability of TAC to induce hypertrophy in CaV3.2 KO appeared to be attributable to the fact that Cn-mediated nuclear NFAT translocation, which is known to induce pathological hypertrophy,12 was not activated in these animals. Surprisingly, the fetal gene program activated with pathological hypertrophy was induced by TAC in CaV3.2 KO without left ventricular hypertrophy.
These are provocative results that, if confirmed, will change thinking in the field. These results suggest that most if not all of NFAT mediated pathological hypertrophy is induced by a very small influx of Ca2+ through reexpressed α1H TTCCs. These new findings also suggest that Cn-NFAT signaling is not influenced by changes in the amplitude and duration of the systolic [Ca2+] transient (contractile [Ca2+]). Contractility in CaV3.2 KO mice after TAC must be greater than in controls which develop left ventricular hypertrophy, because CaV3.2 KO hearts are generating high pressures with less cardiac mass. Therefore, the systolic Ca2+ must be greater in CaV3.2 KO TAC myocytes than in control TAC hearts, yet there was no activation of Cn-NFAT signaling. These results are different from those that have linked the activation of Cn-NFAT signaling with increases in either the rate or amplitude of the cytoplasmic (contractile) [Ca2+] transient in skeletal13 and cardiac muscle.2,14
The report by Chiang et al4 also suggests that Ca2+ activated Cn-NFAT signaling does not play a role in the activation of the fetal gene program after TAC. Their studies show no activation of Cn-NFAT signaling in CaV3.2 KO animals after TAC, but the fetal gene program was induced. In fact, the induction was greater than in controls after TAC. These results suggest that NFAT nuclear translocation has no role in the activation of these well studied fetal genes. Such results are in stark contract to studies that have shown equally convincing data documenting that block of NFAT nuclear translocation eliminates agonist and pressure overload induced hypertrophy and the activation of the fetal gene program.12 Because these data sets seem mutually exclusive this topic clearly needs additional study.
The provocative study by Chiang et al4 suggests that pressure overload causes hypertrophy by inducing the expression of CaV3.2 TTCCs. A very small Ca2+ influx through these channels would need to enter a specialized subsarcolemmal signaling domain that is not influenced by large changes in contractile [Ca2+], where it exclusively activates Cn-NFAT signaling cascades. These new results suggest that pathological hypertrophy is induced through a highly specialized signaling [Ca2+] microdomain that protects Cn-NFAT signaling from changes in contractile Ca2+ and causes pathological hypertrophy without activation of the fetal gene program. These results also exclude a role for TRPC, IP3R, and L-type Ca2+ channels (LTCCs) as a source of Ca2+ regulating cardiac hypertrophy and Cn-NFAT activity, in contrast to numerous reports.2
The second Ca2+ centric report in this issue of Circulation Research, by Terentyev et al,5 identified a novel role for miR-1 in the regulation of contractile Ca2+. Increasing miR-1 in cardiac myocytes caused alterations in the properties of the systolic Ca2+ transient, sarcoplasmic reticulum (SR) Ca2+ loading, and spontaneous and evoked SR Ca2+ release. When myocytes were exposed to catecholamines (isoproterenol [ISO]), only miR-1 myocytes demonstrated arrhythmogenic Ca2+ release. These results suggest that when miR-1 is increased in the diseased heart, catecholamine stress could induce life-threatening arrhythmias.
A novel aspect of this study was that the authors identified that miR-1 targets a regulatory subunit (B56α) of protein phosphatase (PP)2A, leading to reduced PP2A activity and increased phosphorylation of PP2A target proteins. Interestingly, only the phosphorylation state of specific Ca2+/calmodulin kinase (CaMK)II phosphorylation sites were increased in miR-1 myocytes, and inhibition of CaMKII with KN93 reversed dysregulated Ca2+ handling. These results add to the growing body of work linking persistent activation of CaMKII to cardiac dysfunction.15 The authors concluded that hyperphosphorylation of the SR Ca2+ release channel (ryanodine receptor [RyR]) at a known CaMKII site (S2814) alters RyR function and is responsible for arrhythmogenic SR Ca2+ release in the presence of catecholamines (Figure).
The idea that either protein kinase (PK)A or CaMKII mediated phosphorylation of RyR can induce SR Ca2+ leak and cardiac arrhythmias is a contentious topic16 and, in my view, this new study does not resolve critical issues. Although the authors have shown dysregulated Ca2+ in miR-1 myocytes as well as alterations in RyR phosphorylation at RyR S2814, a cause and effect relationship between these 2 miR-1 effects was not proven.
The effects of miR-1 on myocyte Ca2+ handling were complex and varied with conditions. In quiescent miR-1 myocytes, RyR S2814 and LTCC phosphorylation were increased, spark activity (an index of RyR activity) was enhanced and SR Ca2+ loading was reduced. The authors conclude that RyR phosphorylation at S2814 enhances RyR opening to cause diastolic SR Ca2+ “leak,” which reduces SR Ca2+ loading. In voltage-clamped myocytes L-type Ca2+ current and Ca2+ transient amplitude were increased at positive potentials, suggestive of increased excitation-contraction coupling gain. ISO failed to further increase L-type Ca2+ current and the amplitude of the Ca2+ transient in miR-1 myocytes did not increase and was smaller than in controls. miR-1 myocytes field stimulated at 1 Hz had systolic Ca2+ transients that were much larger than in controls and SR Ca2+ loading was now normalized. Why increases in RyR phosphorylation at S2814 would unload the SR in voltage-clamped and quiescent myocytes and maintain SR Ca2+ load when these myocytes are paced is unclear, and suggest other unmeasured factors contribute to miR-1 effects on myocyte contractile Ca2+. Like most new findings, there are many issues to be resolved in future studies.
ISO induced arrhythmogenic Ca2+ release only in miR-1 myocytes. The authors conclude that this resulted from hyperphosphorylation of RyR at S2814. To me, this conclusion is not fully justified. RyR S2814 phosphorylation is increased in miR-1 myocytes under control conditions and arrhythmogenic Ca2+ release is not present. This suggests that CaMKII-mediated phosphorylation of RyR at S2814 is not sufficient to induce single cell arrhythmias. Adding ISO to miR-1 cells induced arrhythmias but did not cause further increases in RyR S2814 phosphorylation or the phosphorylation of the LTCCs, so it is unclear how phosphorylation at S2814 alone can be responsible for the induction of arrhythmias. ISO increased PKA-mediated phosphorylation of RyR at S2808, suggesting that hyperphosphorylation of RyR at this site could be the factor that precipitates arrhythmogenic Ca2+ signaling. However, after inhibition of CaMKII with Kn93, RyR S2814 phosphorylation was reduced, RyR S2808 remained hyperphosphorylated, and arrhythmogenic Ca2+ transients were eliminated. These observations suggest that hyperphosphorylation of RyR at S2808 also is not sufficient to induce Ca2+ release mediated arrhythmias in miR-1 myocytes. Therefore, neither CaMKII phosphorylation of RyR S2814 nor PKA phosphorylation of RyR S2808 alone appear to be sufficient to produce the alterations in RyR behavior that underlie arrhythmogenic SR Ca2+ release. Hyperphosphorylation of both RyR S2814 and S2808 appear to be necessary for this process. Fortunately the model systems to test these ideas are available and hopefully these issues can be resolved.
In summary, 2 new articles in this issue of Circulation Research have identified novel mechanisms for inducing pathological hypertrophy and arrhythmias in cardiac myocytes by altering signaling and contractile Ca2+. New studies will need to confirm these results and determine whether the responsible molecules are good targets for novel therapies for pathological cardiac hypertrophy and arrhythmias.
Sources of Funding
Supported by NIH grant HL33920.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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