Enhanced Galphaq signaling: a common pathway mediates cardiac hypertrophy and apoptotic heart failure

Abstract

Receptor-mediated Gq signaling promotes hypertrophic growth of cultured neonatal rat cardiac myocytes and is postulated to transduce in vivo cardiac pressure overload hypertrophy. Although initially compensatory, hypertrophy can proceed by unknown mechanisms to cardiac failure. We used adenoviral infection and transgenic overexpression of the alpha subunit of Gq to autonomously activate Gq signaling in cardiomyocytes. In cultured cardiac myocytes, overexpression of wild-type Galphaq resulted in hypertrophic growth. Strikingly, expression of a constitutively activated mutant of Galphaq, which further increased Gq signaling, produced initial hypertrophy, which rapidly progressed to apoptotic cardiomyocyte death. This paradigm was recapitulated during pregnancy in Galphaq overexpressing mice and in transgenic mice expressing high levels of wild-type Galphaq. The consequence of cardiomyocyte apoptosis was a transition from compensated hypertrophy to a rapidly progressive and lethal cardiomyopathy. Progression from hypertrophy to apoptosis in vitro and in vivo was coincident with activation of p38 and Jun kinases. These data suggest a mechanism in which moderate levels of Gq signaling stimulate cardiac hypertrophy whereas high level Gq activation results in cardiomyocyte apoptosis. The identification of a single biochemical stimulus regulating cardiomyocyte growth and death suggests a plausible mechanism for the progression of compensated hypertrophy to decompensated heart failure.

Figure 1

Adenovirus-mediated Gαq expression and signaling in cultured cardiac myocytes. (a) Expression of Gαq in neonatal rat cardiac myocytes (representative Western blot of three separate experiments). (b) Overexpression of Gαq stimulates phosphoinositide hydrolysis. P < 0.01 (∗) compared with LacZ; P < 0.001 (#) compared with Gαq WT (n = 9).

Figure 2

Stimulation of myocyte hypertrophy and death by Gαq overexpression. Myocytes infected with indicated adenovirus constructs were fixed, permeabilized, and stained with rhodamine-conjugated phalloidin or polyclonal ANF antiserum. Myocyte hypertrophy is evidenced by increased ANF expression, increased sarcomeric structure, and increased cell size in Gαq-expressing cells at 8 hr. At 24 hr, Q209L expressing cells show shrinkage and death.

Figure 3

Constitutive activation of Gαq signaling causes apoptosis in myocytes. (a) In situ DNA end labeling (ISEL) of myocytes infected with indicated adenovirus constructs. ISEL positive myocytes have reddish brown nuclei. (b) Quantitative analysis of ISEL studies (n = 200 cells per experimental group from three separate experiments). P < 0.001 (∗) compared with LacZ; P < 0.001 (#) compared with Gαq WT. (c) DNA was isolated from myocytes 48 hr after infection with the indicated adenovirus constructs. DNA fragmentation was detected by ethidium bromide fluorescence.

Figure 4

Characteristics of peripartum cardiomyopathy in Gαq-overexpressing mice. (a) Survival curve relating mortality to number of pregnancies. (Inset) Chronology of heart failure development relative to parturition defined as within 7 days of expected delivery (antepartum), within 7 days after delivery (early postpartum), or 7–14 days after delivery (delayed post). (b) Gross morphology (4×) of peripartum nontransgenic (Left) and Gαq-overexpressing (Right) mouse hearts showing cardiomyopathic dilatation of cardiac chambers (LV, left ventricle; RV, right ventricle) with mural thrombi in atria (arrows). (c) Trichrome stain (400×) of hearts depicted in b shows interstitial fibrosis and myocyte replacement (blue-stained cells), without inflammation in transgenic heart (Right).

Figure 5

DNA strand breaks in cardiac myocyte nuclei of Gαq peripartal hearts. (a) Low power (200×) and (c) high power (1,000×) images showing nuclear DNA labeling visualized by fluorescein fluorescence. (b) a counterstained with propidium iodide (red). Apoptotic nuclei appear green or yellow (arrows), occur only in cardiac myocytes, and demonstrate varying degrees of condensed nuclear chromatin (arrows in c). Normal myocyte nuclei are red with a diffuse chromatin staining pattern. (d) DNA laddering of Gαq cardiac DNA. MW, DNA size markers.

Figure 6

JNK and p38 MAP kinase activities in Gαq-overexpressing cardiomyocytes (a) and transgenic mice (b). There is coactivation of both kinases in apoptotic Q209L expressing cardiomyocytes and peripartum (Gαq-25/P) cardiomyopathic mice. P < 0.05 (∗) compared with control (LacZ infected) myocytes or control nontransgenic (NTG) mice, and P < 0.01 (#) compared with control (LacZ, NTG) or Gαq WT (Gαq WT, Gαq-25) groups (n = 6–9 determinations).