Original article
Calcium influx through Cav1.2 is a proximal signal for pathological cardiomyocyte hypertrophy

https://doi.org/10.1016/j.yjmcc.2010.11.012Get rights and content

Abstract

Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca2+) is implicated in hypertrophic signaling pathways, the specific role of Ca2+ influx through the L-type Ca2+ channel (ICa-L) has been controversial and is the topic of this study. To determine if and how sustained increases in ICa-L induce PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca2+ channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP were used. In vivo, β2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume, and cross-sectional area and the expression of PCH markers in a time- and dose-dependent manner. PCH was associated with a hypercontractile phenotype including enhanced ICa-L, fractional shortening, peak Ca2+ transient, at the myocyte level, greater ejection fraction, and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of β2a in cultured AFVMs increased ICa-L, cell volume, protein synthesis, NFAT, and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of ICa-L, intracellular Ca2+, calcineurin, CaMKII, and SERCA. In conclusion, increasing ICa-L is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca2+ pools were shown to be involved.

Research Highlights

► The higher the expression of Cavβ2a in mice, the more Ca2+ influx and hypertrophy. ► Increased Ca2+ influx promotes hypertrophic response to TAC in mice. ► Cavβ2a expression induces cardiac myocyte hypertrophy in culture. ► Increased cytosolic Ca2+ activates calcineurin/NFAT pathway. ► SR-nuclear envelope Ca2+ release activates CaMK II/HDAC pathway.

Introduction

Pathological cardiac hypertrophy (PCH) is an independent risk factor for myocardial infarction, arrhythmia, and subsequent heart failure [1]. It occurs in response to hemodynamic stress such as hypertension, myocardial infarction (MI), and valvular diseases [1]. Pathological cardiovascular stress increases the contractility demands of the heart and its resident myocytes, which is achieved by activating the sympathetic nervous system [2]. Sympathetic neurohormones activate protein kinas A (PKA) to increase Ca2+ influx, SR Ca2+ uptake, storage, and release to increase the amplitude of the systolic Ca2+ transients and contractility [3]. Persistent activation of these signaling pathways also activates Ca2+/calmodulin-dependent kinases (CaMK), which is associated with PCH [4].

Ca2+ regulates many hypertrophic pathways, and well-known examples are the Ca2+-regulated calcineurin/NFAT and CaMK/HDAC pathways [1]. However, the proximal source of Ca2+ that induces PCH is still not well understood. Ca2+ influxes through the Cav1.2/L-type Ca2+ channels (ICa-L) [5], [6], [7], Cav3.2/α1 H T-type Ca2+ channels [8], and transient receptor potential channels (TRPC) [9] have all been proposed to contribute to the pool of Ca2+ that activates hypertrophic pathways. In cardiac myocytes, ICa-L is the major Ca2+ influx and under physiological condition, ICa-L does not activate PCH. Under pathological conditions, activated neurohumoral systems increase ICa-L, which is a likely source of Ca2+ to regulate hypertrophic signaling in vivo. This idea is supported by those studies that have shown a necessary role of enhanced ICa-L for the myocyte hypertrophy induced by phenylephrine (PE) [10], endothelin-1 (ET-1) [11], isoproterenol [12], angiotensin II [9], elevated extracellular KCl [13], and stretch [14]. ICa-L is also able to activate key hypertrophic signaling molecules such as PKC [15] in cardiomyocytes. Cav1.2 channel blockers have been shown to reduce cardiac hypertrophy [6], [16] but the exact mechanism is not clear. More recently, it has been shown that reducing the expression of the Cavβ gene decreases ICa-L and blunts hypertrophy induced by transverse aortic constriction (TAC) in adult rats [10]. We have also shown that Cavβ2a overexpression leads to cardiac hypertrophy at the age of 4 months when heart failure phenotype is present in the HE mice [17]. Other Ca2+ influx pathways also seem to be a source of hypertrophic Ca2+, since the loss of Cav3.2/α1H [8] or TRPCs [18] blunts cardiac hypertrophy induced by TAC. Therefore, different routes of Ca2+ influx may synergically serve as the source for myocyte hypertrophy [19]. The fact that Cav3.1/α1G overexpression in the mice is antihypertrophic rather than prohypertrophic shows the complex nature of Ca2+-mediated induction of PCH.

We used transgenic mice with cardiac-specific overexpression of the β2asubunit of the L-type Ca2+ channel and cultured adult feline ventricular myocytes (AFVM) and neonatal rat ventricular myocytes (NRVM) with enhanced ICa-L by overexpressing the β2a subunit to (1) determine whether increased ICa-L was sufficient to induce myocyte hypertrophy, (2) test if enhanced ICa-L could exacerbate PCH induced by TAC, and (3) determine the signaling cascades for myocyte hypertrophy induced by enhanced ICa-L. Our results show that increasing ICa-L is sufficient to induce myocyte hypertrophy by activation of the calcineurin/NFAT and CaMKII/HDAC signaling pathways. Both cytosolic and SR/ER-nuclear envelop Ca2+ pools were shown to be involved.

Section snippets

Transgenic (TG) mice overexpressing Cavβ2a (β2a)

Cardiac myocytes specific (α-MHC promoter) with inducible (tetracycline-activator (tTA)) β2a mouse lines with high (HE) and low expression (LE) levels were established [17], [20]. β2a increases the open probability and membrane trafficking of the pore-forming Cav1.2α1c subunit. Mice with both β2a and tTA transgenes (double transgenic, DTG) and off doxycycline (DOX, a derivative of tetracycline) were used as the experimental group and mice with single transgene (STG) or no transgene (wild type,

β2a DTG myocytes have increased ICa-L and are hypercontractile

To determine if persistent increases in ICa-L can induce PCH in vivo, we established DTG lines with low (LE, 3.1-fold) and high (HE, 7.4-fold) β2a expression [17]. Previously we found that the expression of β2a in our DTG mice is stable at about 4 months in LE and at 3 months in HE mice, when animals were off DOX at weaning (3 weeks) [17]. ICa-L, myocyte fractional shortening, and Ca2+ transients were measured in myocytes isolated from 4 months LE or 3 months HE mice. ICa-L density was greater in

Discussion

Increases in myocyte [Ca2+] induce PCH [8], [13], [25], but the source of the hypertrophic [Ca2+] is still not clearly defined. The conundrum is that myocyte cytosolic [Ca2+] fluctuates over a wide range during each normal heart beat and can be increased with physiological stimuli such as exercise and pregnancy without inducing PCH [1]. In the present study, we tested the idea that persistent increases in ICa-L, the primary Ca2+ influx pathway in the heart, are sufficient to cause PCH. Our

Conclusion

Our studies suggest that excessive Ca2+ influx through Cav1.2 is a proximal source of Ca2+ for pathological hypertrophy.

Funding

This work was supported by grants from the NIH (HL089312 to S.R.H. and HL088243 to X.C.); HHMI (to J.D.M.) and American Heart Association (AHA0730347N to X.C.).

Disclosures

None.

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