A cardiac-null mutation of Prdm16 causes hypotension in mice with cardiac hypertrophy via increased nitric oxide synthase 1

Abstract

Hypertension or hypotension prevails as a comorbidity in patients with heart failure (HF). Although blood pressure (BP) is an important factor in managing the mortality of HF, the molecular mechanisms of changes in BP have not been clearly understood in cases of HF. We and others have demonstrated that a loss in PRDM16 causes hypertrophic cardiomyopathy, leading to HF. We aimed to determine whether BP is altered in mice that experience cardiac loss of Prdm16 and identify the underlying mechanism of BP-associated changes. BP decreased significantly only in female mice with a cardiac-null mutation of Prdm16 compared with controls, by an invasive protocol under anesthesia and by telemetric method during conscious, unrestrained status. Mice with a cardiac loss of Prdm16 had higher heart-to-body weight ratios and upregulated atrial natriuretic peptide, suggesting cardiac hypertrophy. Plasma aldosterone-to-renin activity ratios and plasma sodium levels decreased in Prdm16-deficient mice versus control. By RNA-seq and in subsequent functional analyses, Prdm16-null hearts were enriched in factors that regulate BP, including Adra1a, Nos1, Nppa, and Nppb. The inhibition of nitric oxide synthase 1 (NOS1) reverted the decrease in BP in cardiac-specific Prdm16 knockout mice. Mice with cardiac deficiency of Prdm16 present with hypotension and cardiac hypertrophy. Further, our findings suggest that the increased expression of NOS1 causes hypotension in mice with a cardiac-null mutation of Prdm16. These results provide novel insights into the molecular mechanisms of hypotension in subjects with HF and contribute to our understanding of how hypotension develops in patients with HF.

Fig 1. Decreased BP in female mice with cardiac-specific inactivation of Prdm16.

(A) BP was measured intra-arterially under anesthesia separately in 5-month-old male and female control (+/+, Prdm16^+/+; Myh6-Cre) and cardiac-specific knockout mice of Prdm16 (CKO, Prdm16^flox/flox; Myh6-Cre), termed Prdm16^CKO. (B) SBP, DBP, and heart rate were measured via a telemetric implant for 24 hours (day and night) during conscious, unrestrained status 1 week after implantation. Error bars, mean ± SEM; number (N), number of mice; statistical significance by student’s t-test, a (P < 0.05), b (P < 0.01), and c (P < 0.005).

Fig 2. Cardiac hypertrophy in mice with cardiac deletion of Prdm16.

(A) Shapes of whole hearts from 5-month-old female mice. (B) Body weight, (C) heart weight, and (D) heart-to-body weight ratio. (E) ANP levels in left ventricle and (F) plasma. Error bars, mean ± SEM; number (N), number of mice; statistical significance by student’s t-test, a (P < 0.05), b (P < 0.01), and c (P < 0.005).

Fig 3. Changes in the renin-angiotensin-aldosterone system in female mice with cardiac-specific null mutation of Prdm16.

(A) Aldosterone levels, (B) renin activity, and (C) aldosterone-to-renin activity ratio (ARR) in plasma. (D) mRNA levels in liver, lung, and kidney for components of the renin-angiotensin system. Error bars, mean ± SEM; number (N), number of mice; statistical significance by student’s t-test, a (P < 0.05), b (P < 0.01), and c (P < 0.005).

Fig 4. Changes in electrolyte balance in cardiac-specific Prdm16 knockout, female mice.

(Ai) Kidney-to-body weight ratio. (Aii) Creatinine clearance rate (CCR). (Aiii) Urea nitrogen in the plasma. (B) 24-hour urine volume. (Ci) Plasma sodium levels. (Cii) Plasma potassium levels. (Ciii) Plasma chloride levels. (Di) Urinary sodium levels. (Dii) Urinary potassium levels. (Diii) Urinary chloride levels. Error bars, mean ± SEM; number (N), number of mice; statistical significance by student’s t-test, b (P < 0.01).

Fig 5. Altered transcriptional profiles in mouse hearts with a Prdm16 deletion and analysis of functional pathways.

RNA-seq analysis in 1-month-old male mouse left ventricular tissue, showing 772 differentially expressed genes (DEGs)—485 upregulated and 287 downregulated—in Prdm16^CKO versus wild-type hearts. (A) PANTHER gene ontology (GO) analysis was used for functional enrichment. Of 772 DEGs, 112 genes were categorized into 17 specific GO classes with significantly functional expression patterns (FDR P-value < 0.05, fold-enrichment > 2). The GO term “Regulation of blood pressure” ranked highest in fold-enrichment, with 4 genes (Adra1a, Nos1, Nppa, and Nppb) included. (B) Comparison of RNA-seq results and qRT-PCR for 4 genes in 1-month-old male mouse left ventricular tissue. (C) qRT-PCR of 4 genes in 5-month-old mouse left ventricular tissue in female Prdm16^CKO versus control. Error bars, mean ± SEM; number (N), number of mice; statistical significance by student’s t-test, c (P < 0.005).

Fig 6. Pharmacological restoration of hypotension in mice with cardiac deficiency of Prdm16.

(A) SBP and heart rate were measured via a telemetric implant in 5-month-old female Prdm16^CKO and control mice after vehicle (saline, except for atropine) or pharmacological administration. (B-C) NOS1 and NOS3, as determined by western blot, in left ventricular tissue from female Prdm16^CKO and control mice. (D) Renal mRNA levels of Nos1, Nos2, and Nos3. PE, phenylephrine; Atro, atropine; Iso, isoprenaline; 7-NI, 7-nitroindazole; and NAME, L-NAME. Error bars, mean ± SEM; number (N), number of mice; statistical significance by student’s t-test, a (P < 0.05) and c (P < 0.005 control versus Prdm16^CKO), * (P < 0.01), and ** (P < 0.005 vehicle versus drug treatment for each genotype).