Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on beta-adrenergic versus Angiotensin II (Ang II)-dependent (G(s) vs. G(alphaq) mediated) modulation of Ca(2+) (i)-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca(2+) currents and Ca(2+) (i) transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca(2+) currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca(2+)/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, beta-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca(2+)-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca(2+) (i)-dependent hypertrophic growth response to Ang II, but not to beta-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT(1) signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for beta-adrenergic Ca(2+) (i)-stimulation in adult myocytes.

Cardiac regeneration is one of the prime visions in cardiovascular research. The mouse neonatal apical resection and left anterior descending artery (LAD) ligation model introduced novel in vivo mammalian assays to study cardiac regeneration. However, recent reports and editorials discussed and critically questioned the value and technical reproducibility of the mouse neonatal myocardial infarction approach, making it paramount to develop and use a reproducible model system. We established a mouse neonatal myocardial infarction model by visually confirmed ligation of the LAD using microsurgery. TdT-mediated dUTP nick-end labeling (TUNEL) proved reproducible massive myocardial infarctions in a defined region of the apex and anterior wall of neonatal and 7-day-old mice. Whereas hearts ligated on postnatal day 7 displayed chronic injury, cardiac samples ligated immediately after birth always showed complete structural regeneration after long-term follow-up. Cardiac regeneration was observed in all mouse stains (C57BL/6J, ICR, and mixed background C57BL/6JxSv129) tested so far. We present a detailed in vivo protocol to study complex mechanisms of complete cardiac repair following ischemic cardiac damage. Neonatal LAD ligation surgery is feasible, and results in reproducible myocardial infarctions 24 h after ligation, and no structural myocardial defects are detectable following long-term follow-up. We encourage the cardiovascular community to use our protocol and teaching video to answer key scientific questions in the field of cardiac regeneration.