Hypertension is an underlying risk factor for cardiovascular disease. Despite this, its pathogenesis remains unknown in most cases. Recently, the transient receptor potential (TRP) channel family was associated with the development of several cardiovascular diseases linked to hypertension. The melastatin TRP channels TRPM4 and TRPM5 have distinct properties within the TRP channel family: they form nonselective cation channels activated by intracellular calcium ions. Here we report the identification of TRPM4 proteins in endothelial cells, heart, kidney, and chromaffin cells from the adrenal gland, suggesting that they have a role in the cardiovascular system. Consistent with this hypothesis, Trpm4 gene deletion in mice altered long-term regulation of blood pressure toward hypertensive levels. No changes in locomotor activity, renin-angiotensin system function, electrolyte and fluid balance, vascular contractility, and cardiac contractility under basal conditions were observed. By contrast, inhibition of ganglionic transmission with either hexamethonium or prazosin abolished the difference in blood pressure between Trpm4^–/– and wild-type mice. Strikingly, plasma epinephrine concentration as well as urinary excretion of catecholamine metabolites were substantially elevated in Trpm4^–/– mice. In freshly isolated chromaffin cells, lack of TRPM4 was shown to cause markedly more acetylcholine-induced exocytotic release events, while neither cytosolic calcium concentration, size, nor density of vesicles were different. We therefore conclude that TRPM4 proteins limit catecholamine release from chromaffin cells and that this contributes to increased sympathetic tone and hypertension.

Pathological cardiac hypertrophy is a major predictor for the development of cardiac diseases. It is associated with chronic neurohumoral stimulation and with altered cardiac Ca²⁺ signalling in cardiomyocytes. TRPC proteins form agonist-induced cation channels, but their functional role for Ca²⁺ homeostasis in cardiomyocytes during fast cytosolic Ca²⁺ cycling and neurohumoral stimulation leading to hypertrophy is unknown. In a systematic analysis of multiple knockout mice using fluorescence imaging of electrically paced adult ventricular cardiomyocytes and Mn²⁺-quench microfluorimetry, we identified a background Ca²⁺ entry (BGCE) pathway that critically depends on TRPC1/C4 proteins but not others such as TRPC3/C6. Reduction of BGCE in TRPC1/C4-deficient cardiomyocytes lowers diastolic and systolic Ca²⁺ concentrations both, under basal conditions and under neurohumoral stimulation without affecting cardiac contractility measured in isolated hearts and in vivo. Neurohumoral-induced cardiac hypertrophy as well as the expression of foetal genes (ANP, BNP) and genes regulated by Ca²⁺-dependent signalling (RCAN1-4, myomaxin) was reduced in TRPC1/C4 knockout (DKO), but not in TRPC1- or TRPC4-single knockout mice. Pressure overload-induced hypertrophy and interstitial fibrosis were both ameliorated in TRPC1/C4-DKO mice, whereas they did not show alterations in other cardiovascular parameters contributing to systemic neurohumoral-induced hypertrophy such as renin secretion and blood pressure. The constitutively active TRPC1/C4-dependent BGCE fine-tunes Ca²⁺ cycling in beating adult cardiomyocytes. TRPC1/C4-gene inactivation protects against development of maladaptive cardiac remodelling without altering cardiac or extracardiac functions contributing to this pathogenesis.

Blood platelet aggregation must be tightly controlled to promote clotting at injury sites but avoid inappropriate occlusion of blood vessels. Thrombin, which cleaves and activates G_q-coupled protease-activated receptors, and collagen-related peptide, which activates the receptor glycoprotein VI, stimulate platelets to aggregate and form thrombi. Coincident activation by these two agonists synergizes, causing the exposure of phosphatidylserine on the cell surface, which is a marker of cell death in many cell types. Phosphatidylserine exposure is also essential to produce additional thrombin on platelet surfaces, which contributes to thrombosis. We found that activation of either thrombin receptors or glycoprotein VI alone produced a calcium signal that was largely dependent only on store-operated Ca²⁺ entry. In contrast, experiments with platelets from knockout mice showed that the presence of both ligands activated nonselective cation channels of the transient receptor potential C (TRPC) family, TRPC3 and TRPC6. These channels principally allowed entry of Na⁺, which coupled to reverse-mode Na⁺/Ca²⁺ exchange to allow calcium influx and thereby contribute to Ca²⁺ signaling and phosphatidylserine exposure. Thus, TRPC channels act as coincidence detectors to coordinate responses to multiple signals in cells, thereby indirectly mediating in platelets an increase in intracellular calcium concentrations and exposure of prothrombotic phosphatidylserine.

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 β-adrenergic versus Angiotensin II (Ang II)-dependent (G_s vs. G_αq mediated) modulation of Ca²⁺ _i-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca²⁺ currents and Ca²⁺ _i transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca²⁺ 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 Ca²⁺/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, β-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca²⁺-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 Ca²⁺ _i-dependent hypertrophic growth response to Ang II, but not to β-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT₁ signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for β-adrenergic Ca²⁺ _i-stimulation in adult myocytes.

The major L-type voltage-gated calcium channels in heart consist of an alpha 1C (Ca(V)1.2) subunit usually associated with an auxiliary beta subunit (Ca-V beta 2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of Ca-V beta 2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra-and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the Ca-V beta 2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, Ca-V beta 2 protein expression declined by > 96% in isolated cardiac myocytes and by > 74% in protein fractions from heart. These latter protein fractions include Ca-V beta 2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other Ca-V beta proteins or changes of Ca(V)1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by < 29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, Ca-V beta 2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, Ca-V beta 2 down-regulation in cardiomyocytes is well tolerated by the adult mice.