Original articleCalcium influx through Cav1.2 is a proximal signal for pathological cardiomyocyte hypertrophy
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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These two authors contribute equally to this study.